Carrier-Antibody Compositions and Methods of Making and Using the Same

ABSTRACT

Described herein are compositions of antibodies and carrier proteins and methods of making and using the same, in particular, as a cancer therapeutic. Also described are lyophilized compositions of antibodies and carrier proteins and methods of making and using the same, in particular, as a cancer therapeutic.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT Application No.PCT/US15/54295, filed Oct. 6, 2015, which claims the benefit of thefiling date of U.S. Provisional Patent Application No. 62/060,484, filedOct. 6, 2014; and U.S. Provisional Patent Application Nos. 62/206,770;62/206,771; and 62/206,772 filed Aug. 18, 2015. The foregoing areincorporated by reference in their entireties.

FIELD OF THE INVENTION

This disclosure relates to novel compositions of antibodies and carrierproteins and methods of making and using the same, in particular, as acancer therapeutic.

STATE OF THE ART

Chemotherapy remains a mainstay for systemic therapy for many types ofcancer, including melanoma. Most chemotherapeutics are only slightlyselective to tumor cells, and toxicity to healthy proliferating cellscan be high (Allen T M. (2002) Cancer 2:750-763), often requiring dosereduction and even discontinuation of treatment. In theory, one way toovercome chemotherapy toxicity issues as well as improve drug efficacyis to target the chemotherapy drug to the tumor using antibodies thatare specific for proteins selectively expressed (or overexpressed) bytumors cells to attract targeted drugs to the tumor, thereby alteringthe biodistribution of the chemotherapy and resulting in more drug goingto the tumor and less affecting healthy tissue. Despite 30 years ofresearch, however, specific targeting rarely succeeds in the therapeuticcontext.

Conventional antibody dependent chemotherapy (ADC) is designed with atoxic agent linked to a targeting antibody via a syntheticprotease-cleavable linker. The efficacy of such ADC therapy is dependenton the ability of the target cell to bind to the antibody, the linker tobe cleaved, and the uptake of the toxic agent into the target cell.Schrama, D. et al. (2006) Nature reviews. Drug discovery 5:147-159.

Antibody-targeted chemotherapy promised advantages over conventionaltherapy because it provides combinations of targeting ability, multiplecytotoxic agents, and improved therapeutic capacity with potentiallyless toxicity. Despite extensive research, clinically effectiveantibody-targeted chemotherapy remains elusive: major hurdles includethe instability of the linkers between the antibody and chemotherapydrug, reduced tumor toxicity of the chemotherapeutic agent when bound tothe antibody, and the inability of the conjugate to bind and enter tumorcells. In addition, these therapies did not allow for control over thesize of the antibody-drug conjugates.

There remains a need in the art for antibody-based cancer therapeuticsthat retain cytoxic effect for targeted drug delivery to providereliable and improved anti-tumor efficacy over prior therapeutics.

In addition, as to any therapeutic application, there also remains aneed for the composition to be stable in its physical, chemical andbiological properties.

Lyophilization, or freeze drying, removes water from a composition. Inthe process, the material to be dried is first frozen and then the iceor frozen solvent is removed by sublimation in a vacuum environment. Anexcipient may be included in pre-lyophilized formulations to enhancestability during the freeze-drying process and/or to improve stabilityof the lyophilized product upon storage. Pikal, M. Biopharm. 3(9)26-30(1990) and Arakawa et al., Pharm. Res. 8(3):285-291 (1991).

While proteins may be lyophilized, the process of lyophilization andreconstitution may affect the properties of the protein. Becauseproteins are larger and more complex than traditional organic andinorganic drugs (i.e. possessing multiple functional groups in additionto complex three-dimensional structures), the formulation of suchproteins poses special problems. For a protein to remain biologicallyactive, a formulation must preserve intact the conformational integrityof at least a core sequence of the protein's amino acids while at thesame time protecting the protein's multiple functional groups fromdegradation. Degradation pathways for proteins can involve chemicalinstability (i.e. any process which involves modification of the proteinby bond formation or cleavage resulting in a new chemical entity) orphysical instability (i.e. changes in the higher order structure of theprotein). Chemical instability can result from deamidation,racemization, hydrolysis, oxidation, beta elimination or disulfideexchange. Physical instability can result from denaturation.aggregation, precipitation or adsorption, for example. The three mostcommon protein degradation pathways are protein aggregation, deamidationand oxidation. Cleland, et al., Critical Reviews in Therapeutic DrugCarrier Systems 10(4): 307-377 (1993).

In the present invention, the composition comprises nanoparticles whichcontain (a) carrier protein (b) antibody and (c) optionally atherapeutic agent. The antibody is believed to be bound to the carrierprotein through hydrophobic interactions which, by their nature, areweak. The lyophilization and reconstitution of such a composition must,therefore, not only preserve the activity of the individual components,but also their relative relationship in nanoparticle.

Further challenges are imposed because the nanoparticles are used intherapy.

For example, rearrangement of the hydrophobic components in thenanoparticle may be mitigated through covalent bonds between thecomponents. However, such covalent bonds pose challenges for thetherapeutic use of nanoparticles in cancer treatment. The antibody,carrier protein, and additional therapeutic agent typically act atdifferent locations in a tumor and through different mechanisms.Non-covalent bonds permit the components of the nanoparticle todissociate at the tumor. Thus, while a covalent bond may be advantageousfor lyophilization, it may be disadvantageous for therapeutic use.

The size of the nanoparticles, and the distribution of the size, is alsoimportant. The nanoparticles of the invention may behave differentlyaccording to their size. At large sizes, the nanoparticles or theagglomeration of these particles may block blood vessels either of whichcan affect the performance and safety of the composition.

Finally, cryoprotectants and agents that assist in the lyophilizationprocess must be safe and tolerated for therapeutic use.

SUMMARY

In one aspect, provided herein are nanoparticle compositions comprisingnanoparticles wherein each of the nanoparticles comprises a carrierprotein, between about 100 to about 1000 antibodies, and optionally atleast one therapeutic agent, wherein the antibodies are arranged outwardfrom the surface of the nanoparticles and wherein the nanoparticles arecapable of binding to a predetermined epitope in vivo.

When administered intravenously, large particles (e.g. greater than 1μm) are typically disfavored because they can become lodged in themicrovasculature of the lungs. At the same time, larger particles canaccumulate in the tumor or specific organs. See e.g. 20-60 micron glassparticle that is used to inject into the hepatic artery feeding a tumorof the liver, called “therasphere” (in clinical use for liver cancer).

Therefore, for intravenous administration, particles under 1 μm areused. Particles over 1 μm are, more typically, administered directlyinto a tumor (“direct injection”) or into an artery feeding into thesite of the tumor.

In another aspect, provided herein are nanoparticle compositionscomprising nanoparticles wherein each of the nanoparticles comprises acarrier protein that is not albumin, between about 100 to about 100antibodies, preferably about 400 to about 800 antibodies, and optionallyat least one therapeutic agent, wherein the antibodies are arranged onan outside surface of the nanoparticles and wherein the nanoparticlesare capable of binding to a predetermined epitope in vivo. Whennanoparticles multimerize, the number of antibodies is increasedproportionally. For example, if a 160 nm nanoparticle contains 400antibodies, a 320 nm dimer contains about 800 antibodies.

In another aspect, provided herein are nanoparticle compositionscomprising nanoparticles, wherein each of the nanoparticles comprisescarrier protein, between about 400 to about 800 antibodies, andoptionally at least one therapeutic agent that is not paclitaxel,wherein the antibodies are arranged on a surface of the nanoparticlessuch that the binding portion of the antibody is directed outward fromthat surface and wherein the nanoparticles are capable of binding to apredetermined epitope in vivo.

In other embodiments, the nanoparticles multimerize, e.g. dimerize.Multimerization may be observed as multiples of the weight or size ofthe unit molecule, e.g. 160 nm particles multimerize to about 320 nm,480 nm, 640 nm, etc. In some embodiments, less than 20% of thepopulation are multimers. In some embodiments, more than 80% of thepopulation are multimers.

In one embodiment, the weight ratio of carrier-bound drug to antibody(e.g. albumin-bound paclitaxel to bevacizumab) is between about 5:1 toabout 1:1. In one embodiment, the weight ratio of carrier-bound drug toantibody is about 10:4. In one embodiment, the antibody is asubstantially single layer of antibodies on all or part of the surfaceof the nanoparticle. In one embodiment, less than 0.01% of nanoparticlesin the composition have a size selected from the group consisting ofgreater than 200 nm, greater than 300 nm, greater than 400 nm, greaterthan 500 nm, greater than 600 nm, greater than 700 nm and greater than800 nm. Larger sizes are believed to be the result of multimerization ofseveral nanoparticles, each comprising a core and antibody coating onall or part of the surface of each nanoparticle.

The invention further includes lyophilized compositions, and lyophilizedcompositions that do not materially differ from, or are the same as, theproperties of freshly-prepared nanoparticles. In particular, thelypholized composition, upon resuspending in aqueous solution, issimilar or identical to the fresh composition in terms of particle size,particle size distribution, toxicity for cancer cells, antibodyaffinity, and antibody specificity. The invention is directed to thesurprising finding that lyophilized nanoparticles retain the propertiesof freshly-made nanoparticles notwithstanding the presence of twodifferent protein components in these particles.

In one aspect, this invention relates to a lyophilized nanoparticlecomposition comprising nanoparticles, wherein each of the nanoparticlescomprises a carrrier-bound drug core and an amount of antibody arrangedon a surface of the core such that the binding portion of the antibodyis directed outward from that surface, wherein the antibodies retaintheir association with the outside surface of the nanoparticle uponreconstitution with an aqueous solution. In one embodiment, thelyophilized composition is stable at room temperature for at least 3months. In one embodiment, the reconstituted nanoparticles retain theactivity of the therapeutic agent and are capable of binding to thetarget in vivo.

In one embodiment, the average reconstituted nanoparticle size is fromabout 130 nm to about 1 μm. In a preferred embodiment, the averagereconstituted nanoparticle size is from about 130 nm to about 200 nm,and more preferably about 160 nm. In one embodiment, in the averagereconstituted nanoparticle size is from greater than 800 nm to about 3.5μm, comprising multimers of smaller nanoparticles, e.g. multimers of100-200 nm nanoparticles. In one embodiment, the weight ratio of core toantibody is from greater than 1:1 to about 1:3.

In one aspect, this disclosure relates to a lyophilized nanoparticlecomposition comprising nanoparticles, wherein each of the nanoparticlescomprises: (a) an albumin-bound paclitaxel core and (b) between about400 to about 800 molecules of bevacizumab arranged on a surface of thealbumin-bound paclitaxel core such that the binding portion of theantibody is directed outward from that surface, wherein the antibodiesretain their association with the surface of the nanoparticle uponreconstitution with an aqueous solution, provided that said lyophilizedcomposition is stable at about 20° C. to about 25° C. for at least 3months and the reconstituted nanoparticles are capable of binding toVEGF in vivo.

In other aspects, this disclosure relates to a lyophilized nanoparticlecomposition comprising nanoparticles, wherein each of the nanoparticlescomprises: (a) an albumin-bound paclitaxel core and (b) an amount ofbevacizumab arranged on a surface of the albumin-bound paclitaxel coresuch that the binding portion of the antibody is directed outward fromthat surface, wherein the antibodies retain their association with thesurface of the nanoparticle upon reconstitution with an aqueoussolution, provided that said lyophilized composition is stable at about20° C. to about 25° C. for at least 3 months and the reconstitutednanoparticles are capable of binding to VEGF in vivo, and furtherwherein the average reconstituted nanoparticle size is not substantiallydifferent from the particle size of the freshly prepared nanoparticles.In some embodiments, the particle sizes are between 200 and 800 nm,including 200, 300, 400, 500, 600, 700 or 800 nm. In other embodiments,the particles are larger, e.g. from greater than 800 nm to about 3.5 μm.In some embodiments, the particles are multimers of nanoparticles.

In some embodiments, the weight ratio of albumin-bound paclitaxel tobevacizumab is between about 5:1 to about 1:1. In other embodiments, theweight ratio of albumin-bound paclitaxel to bevacizumab is about 10:4.In further embodiments, the weight ratio of albumin-bound paclitaxel tobevacizumab is from greater than 1:1 to about 1:3.

In some embodiments, the core is albumin-bound paclitaxel, and theantibodies are selected from antibodies that selectively recognize VEGF(e.g. bevacizumab/Avastin), antibodies that selectively recognize CD20(e.g. rituximab/Rituxin) and antibodies that selectively recognize Her2(Trastuzumab/Herceptin).

In some embodiments, the at least one therapeutic agent is locatedinside the nanoparticle. In other embodiments, the at least onetherapeutic agent is located on the outside surface of the nanoparticle.In yet other embodiments, the at least one therapeutic agent is locatedinside the nanoparticle and on the outside surface of the nanoparticle.

In some embodiments, the nanoparticle contains more than one type oftherapeutic agent. For example, a taxane and a platinum drug, e.g.paclitaxel and cisplatin.

In some embodiments, the antibodies are selected from the groupconsisting of ado-trastuzumab emtansine, alemtuzumab, bevacizumab,cetuximab, denosumab, dinutuximab, ipilimumab, nivolumab, obinutuzumab,ofatumumab, panitumumab, pembrolizumab, pertuzumab, rituximab, andtrastuzumab. In some embodiments, the antibodies are a substantiallysingle layer of antibodies on all or part of the surface of thenanoparticle.

In further embodiments, the antibodies are less glycosylated thannormally found in natural human antibodies. Such glycosylation can beinfluenced by e.g. the expression system, or the presence ofglycosylation inhibitors during expression. In some embodiments, theglycosylation status of an antibody is altered through enzymatic orchemical action.

In some embodiments, the at least one therapeutic agent is selected fromthe group consisting of abiraterone, bendamustine, bortezomib,carboplatin, cabazitaxel, cisplatin, chlorambucil, dasatinib, docetaxel,doxorubicin, epirubicin, erlotinib, etoposide, everolimus, gefitinib,idarubicin, imatinib, hydroxyurea, imatinib, lapatinib, leuprorelin,melphalan, methotrexate, mitoxantrone, nedaplatin, nilotinib,oxaliplatin, paclitaxel, pazopanib, pemetrexed, picoplatin, romidepsin,satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin,vinblastine, vinorelbine, vincristine, and cyclophosphamide.

In some embodiments, the nanoparticle further comprises at least oneadditional therapeutic agent that is not paclitaxel or bevacizumab.

In some embodiments, the antibodies, carrier protein and, when present,therapeutic agent, are bound through non-covalent bonds.

In some embodiments, the carrier protein is selected from the groupconsisting of gelatin, elastin, gliadin, legumin, zein, a soy protein, amilk protein, and a whey protein. In other embodiments, the carrierprotein is albumin, for example, human serum albumin.

In some embodiments, the composition is formulated for intravenousdelivery. In other embodiments, the composition is formulated for directinjection or perfusion into a tumor.

In some embodiments, the average nanoparticle size in the composition isfrom greater than 800 nm to about 3.5 μm.

In some embodiments, the nanoparticles have a dissociation constantbetween about 1×10⁻¹¹ M and about 1×10⁻⁹ M.

In another aspect, provided herein are methods of making nanoparticlecompositions, wherein said methods comprise contacting the carrierprotein and the optionally at least one therapeutic agent with theantibodies in a solution having a pH of between 5.0 and 7.5 and atemperature between about 5° C. and about 60° C., between about 23° C.and about 60° C., or between about 55° C. and about 60° C. underconditions and ratios of components that will allow for formation of thedesired nanoparticles. In one embodiment, the nanoparticle is made at55-600 C and pH 7.0. In another aspect, provided herein are methods ofmaking the nanoparticle compositions, wherein said method comprises (a)contacting the carrier protein and optionally the at least onetherapeutic agent to form a core and (b) contacting the core with theantibodies in a solution having a pH of about 5.0 to about 7.5 at atemperature between about 5° C. and about 60° C., between about 23° C.and about 60° C., or between about 55° C. and about 60° C. underconditions and ratios of components that will allow for formation of thedesired nanoparticles.

The amount of components (e.g., carrier protein, antibodies, therapeuticagents, combinations thereof) is controlled in order to provide forformation of the desired nanoporaticles. A composistion wherein theamount of components is too dilute will not form the nanoparticles asdesirbed herein. In a prefered embodiment, weight ratio of carrierprotein to antibody is 10:4. In some embodiments, the amount of carrierprotein is between about 1 mg/mL and about 100 mg/mL. In someembodiments, the amount of antibody is between about 1 mg/mL and about30 mg/mL. For example, in some embodiments, the ratio of carrierprotein: antibody: solution is approximately 9 mg of carrier protein(e.g., albumin) to 4 mg of antibody (e.g., BEV) in 1 mL of solution(e.g., saline). An amount of therapeutic agent (e.g., taxol) can also beadded to the carrier protein.

In further embodiments, the nanoparticles are made as above, and thenlyophilized.

In another aspect, provided herein are methods for treating a cancercell, the method comprising contacting the cell with an effective amountof a nanoparticle composition disclosed herein to treat the cancer cell.

In another aspect, provided herein are methods for treating a tumor in apatient in need thereof, the method comprising contacting the cell withan effective amount of a nanoparticle composition disclosed herein totreat the tumor. In some embodiments, the size of the tumor is reduced.In other embodiments, the nanoparticle composition is administeredintravenously. In yet other embodiments, the nanoparticle composition isadministered by direct injection or perfusion into the tumor.

In some embodiments, the methods provided herein include the steps of:a) administering the nanoparticle composition once a week for threeweeks; b) ceasing administration of the nanoparticle composition for oneweek; and c) repeating steps a) and b) as necessary to treat the tumor.

In related embodiments, the treatment comprises administration of thetargeting antibody prior to administration of the nanoparticles. In oneembodiment, the targeting antibody is administered between about 6 and48, or 12 and 48 hours prior to administration of the nanoparticles. Inanother embodiment, the targeting antibody is administered between 6 and12 hours prior to administration of the nanoparticles. In yet anotherembodiment, the targeting antibody is administered between 2 and 8 hoursprior to administration of the nanoparticles. In still otherembodiments, the targeting antibody is administered a week prior toadministration of the nanoparticles. For example, administration of adose of BEV 24 hours prior to administration of AB 160. In anotherexample, prior administration of rituximab prior to administering ARnanoparticles. The antibody administered prior to the nanoparticle maybe administered as a dose that is subtherapeutic, such as ½, 1/10^(th)or 1/20 the amount normally considered therapeutic. Thus, in man,pretreatment with BEV may comprise administration of 1 mg/kg BEV whichis 1/10^(th) the ususual dose, followed by administration of AB160.

In some embodiments, the therapeutically effective amount comprisesabout 75 mg/m² to about 175 mg/m² of the carrier protein (i.e.,milligrams carrier protein per m² of the patient). In other embodiments,the therapeutically effective amount comprises about 75 mg/m² to about175 mg/m² of therapeutic agent (e.g., paclitaxel). In other embodiments,the therapeutically effective amount comprises about 30 mg/m² to about70 mg/m² of the antibody. In yet other embodiments, the therapeuticallyeffective amount comprises about 30 mg/m² to about 70 mg/m² bevacizumab.

In one specific embodiment, the lypholized composition comprises fromabout 75 mg/m² to about 175 mg/m² of the carrier protein which ispreferably albumin; from about 30 mg/m² to about 70 mg/m² of theantibody which is preferably bevacizumab; and from about about 75 mg/m²to about 175 mg/m² of paclitaxel.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are representative only of the invention and arenot intended as a limitation. For the sake of consistency, thenanoparticles of this invention using Abraxane® and bevacizumab employthe acronym “AB” and the number after AB such as AB 160 is meant toconfer the average particle size of these nanoparticles (in nanometers).Likewise, when the antibody is rituximab, the acronym is “AR” while thenumber thereafter remains the same.

FIG. 1A shows flow cytometry scatterplots including: Abraxane®(ABX—commercially available from Celgene Corporation, Summit, N.J.07901) stained with secondary antibody only (top left panel), ABXstained with goat anti-mouse IgG1 Fab plus secondary antibody (top rightpanel), AB160 (which is a nanoparticle of albumin-bound paclitaxel tobevacizumab in a ratio of about 10:4 and have an average particle sizeof 160 nm) stained with secondary antibody only (bottom left panel), orAB 160 stained with goat anti-mouse IgG1 Fab plus secondary antibody(bottom right panel).

FIG. 1B shows a representative electron micrograph after incubation ofAB160 with gold particle-labeled anti-human IgG Fe.

FIG. 1C shows a pie chart (top) indicating the percentages of totalpaclitaxel in AB160 fractions (particulate, proteins greater than 100 kDand proteins less than 100 kD); and a Western blot with antibodiesagainst mouse IgG Fab (BEV) and paclitaxel to verify co-localization(bottom).

FIG. 1 D represents the activity of paclitaxel in an in vitro toxicityassay with A375 human melanoma cells, compared to ABX alone. The resultsare represented by the average (+/−SEM) proliferation index, which isthe percentage of total proliferation of untreated cells. This datarepresents 3 experiments and differences were not significant.

FIG. 1E represents results from a VEGF ELISA of supernatant afterco-incubation of VEGF with ABX and AB 160 to determine binding of theligand, VEGF, by the antibody. The results are shown as the averagepercentage +/−SEM of VEGF that was unbound by the 2 complexes. The datarepresents 3 experiments ** P<0.005.

FIG. 2A shows the size of the complexes (determined by light scatteringtechnology) formed by adding BEV (bevacizumab) to ABX under conditionswhere nanoparticles and higher are formed. Increasing concentrations ofBEV (0-25 mg) were added to 10 mg of ABX and the size of the complexesformed was determined. The average size of the complexes (146 nm to 2,166 nm) increased as the concentration of BEV was increased. The data isdisplayed as volume of sample/size and graphs show the size distributionof the particles. This data is representative of 5 separate drugpreparations. As a comparison, ABX, by itself, has an average particlesize of about 130 nm.

FIG. 2B shows affinity of the binding of ABX and BEV (as determined bylight absorption (BLItz) technology). The data is displayed asdissociation constant (Kd). The binding affinity of particles made atfour pH levels (3, 5, 7, 9) and 3 temperatures (RT, 37° C. and 58° C.)was assessed, and the data are representative of 5 experiments.

FIG. 2C shows the stability of the nanoparticle complexes from FIG. 2Bin serum as determined by a nanoparticle tracking analysis (NTA) onNanosight 300 (NS300). The data are displayed as the number ofparticles/mg of ABX and compares AB160 prepared at RT and pH 7 (AB16007;particle size, pH), 58° C. and pH 7 (AB1600758; particle size, pH,temperature) and 58° C. and pH 5 (AB1600558; particle size, pH,temperature), relative to ABX alone under each condition. Once particleswere prepared, they were added to human AB serum for 15, 30, and 60minutes to determine stability in serum over time.

FIG. 3A shows in vivo testing of AB nanoparticles in athymic nude miceinjected with 1×10⁶ A375 human melanoma cells in the right flank andtreated with PBS, 12 mg/kg BEV, 30 mg/kg ABX, 12 mg/kg BEV+30 mg/kg ABX,or AB160 (having about 12 mg/kg BEV and about 30 mg/kg ABX) at tumorsize between approximately 600 mm³ to 900 mm³ Data is represented at day7-post treatment as the percent change in tumor size from baseline (thesize of the tumor on the day of treatment). Student's t-test was used todetermine significance. The p-values for the AB particles were allsignificantly different than PBS, the individual drugs alone and the 2drugs given sequentially.

FIG. 3B shows Kaplan-Meier curves generated for median survival of themice analyzed in FIG. 3A. Significance was determined using theMantle-Cox test comparing survival curves.

FIG. 3C shows the percent change from baseline for mice treated whentumors were less than or greater than 700 mm³, to ascertain whether thesize of the tumor affected tumor response for the ABX only and AB160groups. The Student's t-test was used to determine significance; the ABXonly groups showed no significant difference (p=0.752) based on tumorsize, while the AB160 groups were significantly different (p=0.0057).

FIG. 3D shows in vivo testing of AB nanoparticles in athymic nude miceinjected with 1×10⁶ A375 human melanoma cells in the right flank andtreated with PBS, 30 mg/kg ABX, or 45 mg/kg BEV and AB160, AB580(nanoparticle of albumin-bound paclitaxel to bevacizumab having anaverage particle size of 580 nm) or AB1130 (nanoparticle ofalbumin-bound paclitaxel to bevacizumab having an average particle sizeof 1130 nm) at tumor size between approximately 600 mm³ to 900 mm³. Datais represented at day 7-post treatment as the percent change in tumorsize from baseline (the size of the tumor on the day of treatment).Student's t-test was used to determine significance. The p-values forthe AB particles were all significantly different than PBS, theindividual drugs alone and the 2 drugs given sequentially. Thedifference among the AB particles of different sizes was notsignificant.

FIG. 3E shows Kaplan-Meier curves generated for median survival of themice analyzed in FIG. 3D. Significance was determined using theMantle-Cox test comparing survival curves.

FIG. 4A demonstrates blood paclitaxel concentration displayed in linegraph with y-axis in log scale, based on blood and tumor samples takenfrom non-tumor and tumor bearing mice at 0-24 hours after IV injectionwith 30 mg/kg of paclitaxel in the context of ABX or AB160 and measuredby LC-MS. Mice were IV injected at time 0, with blood samples taken andthe mice sacrificed at time points of 0, 4, 8, 12, and 24 hours. Therewere at least 3 mice per time point. Student's t-test was utilized todetermine if any differences in concentrations between ABX and AB 160were significant.

FIG. 4B demonstrates the blood paclitaxel concentration from FIG. 4A,displayed in line graph with y-axis in numeric scale.

FIG. 4C shows the C_(max), half-life and AUC values calculated from theblood concentration data provide in FIGS. 4A and 4B.

FIG. 4D demonstrates blood paclitaxel concentration displayed in linegraph with y-axis in log scale from a second pharmacokinetic experimentusing earlier time points (2 to 8 hours).

FIG. 4E demonstrates the blood paclitaxel concentration from FIG. 4D,displayed in line graph with y-axis in numeric scale.

FIG. 4F shows blood paclitaxel concentration in mice in which the tumorswere allowed to grow to a larger size before ABX and AB160 injections.

FIG. 4G shows the C^(max) and the AUC calculated from the data in FIG.4F.

FIG. 411 shows paclitaxel concentrations in the tumors from the secondmouse experiment as determined by LC-MS. Data are displayed as μg ofpaclitaxel/mg of tumor tissue. Student's t-test was utilized todetermine if differences were significant.

FIG. 4I shows 1-125 radioactivity levels in mice treated with AB160relative to ABX alone.

FIG. 4J shows a graphical represenatation of the 1-125 radioactivitylevels shown in FIG. 4I.

FIG. 5A shows particle size measurements and affinity of nanoparticlesmade with rituximab. 10 mg/ml of ABX was incubated with rituximab (RIT)at 0-10 mg/ml and light scatter technology (Mastersizer 2000) was usedto determine resulting particle sizes. Data are displayed as the percentvolume of particles at each size and the curves represent particle sizedistributions (top). The table (bottom) shows the sizes of the resultingparticles at each concentration of antibody.

FIG. 5B shows particle size measurements and affinity of nanoparticlesmade with trastuzumab. 10 mg/ml of ABX was incubated with trastuzumab(HER) at 0-22 mg/ml and light scatter technology (Mastersizer 2000) wasused to determine resulting particle sizes. Data are displayed as thepercent volume of particles at each size and the curves representparticle size distributions (top). The table (bottom) shows the sizes ofthe resulting particles at each concentration of antibody.

FIG. 5C shows the binding affinity of rituximab and trastuzumab ascompared to ABX at pH 3, 5, 7 and 9, determined by biolayerinterferometry (BLitz) technology. The dissociation constants aredisplayed for each interaction.

FIG. 6A shows in vitro toxicity of AR160 as tested with theCD20-positive Daudi human lymphoma cell line. The data are displayed ina graph of the proliferation index, which is the percent of FITCpositive cells in treated wells relative to FITC positive cells in theuntreated well (the highest level of proliferation).

FIG. 6B shows in vivo tumor efficacy in athymic nude mice injected with5×10⁶ Daudi human lymphoma cells in the right flank. The tumors wereallowed to grow to 600 mm³ to 900 mm³ and the mice were treated withPBS, 30 mg/kg ABX, 12 mg/kg rituximab, 12 mg/kg rituximab+30 mg/kg ABX,or AR160. Tumor response was determined at day 7 post-treatment by thepercent change in tumor size from the first day of treatment.Significance was determined by Student's t-test; the percent change frombaseline was significantly different between the AR160 treated mice andall other groups (p<0.0001).

FIG. 6C shows Kaplan-Meier survival curves generated from the experimentshown in FIG. 6B. Median survival for each treatment group is shown. AMantle-Cox test was used to determine whether survival curve differenceswere significant.

FIG. 7A demonstrates addition of another chemotherapy drug (cisplatin)cisplatin to AB160. ABX (5 mg/ml) was incubated with cisplatin (0.5mg/ml) at room temperature for 30 minutes and free cisplatin wasmeasured by HPLC in the supernatant after ABX particulate was removed.The quantity of free cisplatin was subtracted from the startingconcentration to determine the quantity of cisplatin that bound to theABX. The data are displayed in a column graph, along with the startingconcentration (cisplatin).

FIG. 7B shows the toxicity of cisplatin-bound ABX (AC) in aproliferation assay of A375 human melanoma cells. After 24 hours of drugexposure and EdU incorporation, the cells were fixed, permeabilized andlabeled with a FITC conjugated anti-EdU antibody. The data is displayedin a graph of the proliferation index, which is the percent of FITCpositive cells in treated wells compared to FITC positive cells in theuntreated well (the highest level of proliferation).

FIG. 7C shows in vivo tumor efficacy of AC (ABC complex; cisplatin-boundABX) in athymic nude mice injected with 1×10⁶ A375 human melanoma cellsin the right flank. The tumors were allowed to grow to 600 mm³ to 900mm³ and the mice were treated with PBS, 30 mg/kg ABX, 2 mg/kg cisplatin,AB160, 2 mg/kg cisplatin+AB160 or ABC160. Tumor response was determinedat day 7 post-treatment by the percent change in tumor size from the dayof treatment. Significance was determined by Student's t-test; thepercent change from baseline was significantly different between the ABC160 treated mice and PBS-, cisplatin-, or ABX-treated mice (p<0.0001).There was no significant difference between the AB160, AB160+cisplatin,and ABC 160 treated groups for day 7 post-treatment percent change frombaseline.

FIG. 7D shows Kaplan-Meier survival curves generated based on theexperiment shown in FIG. 7C and median survival for each treatment groupis shown. A Mantle-Cox test was used to determine whether survival curvedifferences were significant.

FIG. 8A shows the size distribution of AB160 nanoparticles that werelyophilized, stored at room temperature for one month, andreconstituted, as compared to fresh AB 160 or ABX alone.

FIG. 8B shows the ligand (VEGF) binding ability of AB 160 nanoparticlesthat were lyophilized, stored at room temperature for one month, andreconstituted, as compared to fresh AB160 or ABX alone.

FIG. 8C shows in vitro cancer cell toxicity of AB 160 nanoparticles thatwere lyophilized, stored at room temperature for one month, andreconstituted, as compared to fresh AB160 or ABX alone.

FIG. 8 D shows the size distribution of AB160 nanoparticles that werelyophilized, stored at room temperature for ten months, andreconstituted, as compared to fresh AB 160 or ABX alone.

FIG. 8E shows the ligand (VEGF) binding ability of AB160 nanoparticlesthat were lyophilized, stored at room temperature for ten months, andreconstituted, as compared to fresh AB160 or ABX alone.

FIG. 8F shows in vitro cancer cell toxicity of AB 160 nanoparticles thatwere lyophilized, stored at room temperature for ten months, andreconstituted, as compared to fresh AB160 or ABX alone.

FIGS. 9A-C show the size distributions of the ABX-BEV complexes at I.V.infusion conditions (ABX final concentration of 5 mg/mL) incubated insaline at room temperature for up to 24 hours (FIGS. A and B). By 4hours at room temperature, there is some evidence of complex breakdownby ELISA (20%, FIG. C).

FIG. 10 shows in vitro incubation for 30 seconds of ABX (top panel) orAB160 (bottom panel) in saline or heparinized human plasma at relativevolume ratios of 9:1 or 1:1.

FIGS. 11A-E show in vivo testing of athymic nude mice injected with1×10⁶ A375 human melanoma cells in the right flank and treated with(FIG. 11A) PBS, (FIG. 11B) 12 mg/kg BEV, (FIG. 11C) 30 mg/kg ABX, (FIG.11D) AB 160, or (FIG. 11E) pretreated with 01.2 mg/kg BEV and, 24 hrlater, AB 160. Data is represented at day 7-post and 10-day treatment astumor volume in mm³.

FIG. 11F summarizes the day 7-post treatment data from FIGS. 11A-E.

FIG. 11G summarizes the day 10-posttreatment data from FIGS. 11A-E.

DETAILED DESCRIPTION

After reading this description it will become apparent to one skilled inthe art how to implement the invention in various alternativeembodiments and alternative applications. However, all the variousembodiments of the present invention will not be described herein. Itwill be understood that the embodiments presented here are presented byway of an example only, and not limitation. As such, this detaileddescription of various alternative embodiments should not be construedto limit the scope or breadth of the present invention as set forthbelow.

Before the present invention is disclosed and described, it is to beunderstood that the aspects described below are not limited to specificcompositions, methods of preparing such compositions, or uses thereof assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

The detailed description of the invention is divided into varioussections only for the reader's convenience and disclosure found in anysection may be combined with that in another section. Titles orsubtitles may be used in the specification for the convenience of areader, which are not intended to influence the scope of the presentinvention.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In this specification and inthe claims that follow, reference will be made to a number of terms thatshall be defined to have the following meanings:

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The term “about” when used before a numerical designation, e.g.,temperature, time, amount, concentration, and such other, including arange, indicates approximations which may vary by (+) or (−) 10%, 5%,1%, or any subrange or subvalue there between. Preferably, the term“about” when used with regard to a dose amount means that the dose mayvary by +/−10%. For example, “about 400 to about 800 antibodies”indicates that an outside surface of a nanoparticles contain an amountof antibody between 360 and 880 particles.

“Comprising” or “comprises” is intended to mean that the compositionsand methods include the recited elements, but not excluding others.“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination for the stated purpose. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) of the claimed invention.“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.

The term “nanoparticle” as used herein refers to particles having atleast one dimension which is less than 5 microns. In preferredembodiments, such as for intravenous administration, the nanoparticle isless than 1 micron. For direct administration, the nanoparticle islarger. Even larger particles are expressly contemplated by theinvention.

In a population of particles, the size of individual particles aredistributed about a mean. Particle sizes for the population cantherefore be represented by an average, and also by percentiles. D50 isthe particle size below which 50% of the particles fall. 10% ofparticles are smaller than the DIO value and 90% of particles aresmaller than D90. Where unclear, the “average” size is equivalent toD50. So, for example, AB160 refers to nanoparticles having an averagesize of 160 nanometers.

The term “nanoparticle” may also encompass discrete multimers of smallerunit nanoparticles. For example, a 320 nm particle comprises a dimer ofa unit 160 nm nanoparticle. For 160 nm nanoparticles, multimers wouldtherefore be approximately 320 nm, 480 nm, 640 nm, 800 nm, 960 nm, 1120nm, and so on.

The term “carrier protein” as used herein refers to proteins thatfunction to transport antibodies and/or therapeutic agents. Theantibodies of the present disclosure can reversibly bind to the carrierproteins. Exemplary carrier proteins are discussed in more detail below.

The term “core” as used herein refers to central or inner portion of thenanoparticle which may be comprised of a carrier protein, a carrierprotein and a therapeutic agent, or other agents or combination ofagents. In some embodiments, a hydrophobic portion of the antibody maybe incorporated into the core.

The term “therapeutic agent” as used herein means an agent which istherapeutically useful, e.g., an agent for the treatment, remission orattenuation of a disease state, physiological condition, symptoms, oretiological factors, or for the evaluation or diagnosis thereof. Atherapeutic agent may be a chemotherapeutic agent, for example, mitoticinhibitors, topoisomerase inhibitors, steroids, anti-tumor antibiotics,antimetabolites, alkylating agents, enzymes, proteasome inhibitors, orany combination thereof.

The term “antibody” or “antibodies” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules (i.e., molecules that contain an antigenbinding site that immuno-specifically bind an antigen). The term alsorefers to antibodies comprised of two immunoglobulin heavy chains andtwo immunoglobulin light chains as well as a variety of forms includingfull length antibodies and portions thereof; including, for example, animmunoglobulin molecule, a monoclonal antibody, a chimeric antibody, aCDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, aFv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), adiabody, a multispecific antibody, a dual specific antibody, ananti-idiotypic antibody, a bispecific antibody, a functionally activeepitope-binding fragment thereof, bifunctional hybrid antibodies (e.g.,Lanzavecchia et al., Eur. J Immunol. 17, 105 (1987)) and single chains(e.g., Huston et al., Proc. Natl. Acad. Sci. US.A., 85, 5879-5883 (1988)and Bird et al., Science 242, 423-426 (1988), which are incorporatedherein by reference). (See, generally, Hood et al., Immunology,Benjamin, N.Y., 2ND ed. (1984); Harlow and Lane, Antibodies. ALaboratory Manual, Cold Spring Harbor Laboratory (1988); Hunkapiller andHood, Nature, 323, 15-16 (1986), which are incorporated herein byreference). The antibody may be of any type (e.g., IgG, IgA, IgM, IgE orIgD). Preferably, the antibody is IgG. An antibody may be non-human(e.g., from mouse, goat, or any other animal), fully human, humanized,or chimeric.

The term “dissociation constant,” also referred to as “K_(d),” refers toa quantity expressing the extent to which a particular substanceseparates into individual components (e.g., the protein carrier,antibody, and optional therapeutic agent).

The terms “lyophilized,” “lyophilization” and the like as used hereinrefer to a process by which the material (e.g., nanoparticles) to bedried is first frozen and then the ice or frozen solvent is removed bysublimation in a vacuum environment. An excipient is optionally includedin pre-lyophilized formulations to enhance stability of the lyophilizedproduct upon storage. In some embodiments, the nanoparticles can beformed from lyophilized components (carrier protein, antibodiy andoptional therapeutic) prior to use as a therapeutic. In otherembodiments, the carrier protein, antibody, and optional therapeuticagent are first combined into nanoparticles and then lyophilized. Thelyophilized sample may further contain additional excipients.

The term “bulking agents” comprise agents that provide the structure ofthe freeze-dried product. Common examples used for bulking agentsinclude mannitol, glycine, lactose and sucrose. In addition to providinga pharmaceutically elegant cake, bulking agents may also impart usefulqualities in regard to modifying the collapse temperature, providingfreeze-thaw protection, and enhancing the protein stability overlong-term storage. These agents can also serve as tonicity modifiers.

The term “buffer” encompasses those agents which maintain the solutionpH in an acceptable range prior to lyophilization and may includesuccinate (sodium or potassium), histidine, phosphate (sodium orpotassium), Tris(tris(hydroxymethyl)aminomethane), diethanolamine,citrate (sodium) and the like. The buffer of this invention has a pH inthe range from about 5.5 to about 6.5; and preferably has a pH of about6.0. Examples of buffers that will control the pH in this range includesuccinate (such as sodium succinate), gluconate, histidine, citrate andother organic acid buffers.

The term “cryoprotectants” generally includes agents which providestability to the protein against freezing-induced stresses, presumablyby being preferentially excluded from the protein surface. They may alsooffer protection during primary and secondary drying, and long-termproduct storage. Examples are polymers such as dextran and polyethyleneglycol; sugars such as sucrose, glucose, trehalose, and lactose;surfactants such as polysorbates; and amino acids such as glycine,arginine, and serine.

The term “lyoprotectant” includes agents that provide stability to theprotein during the drying or ‘dehydration’ process (primary andsecondary drying cycles), presumably by providing an amorphous glassymatrix and by binding with the protein through hydrogen bonding,replacing the water molecules that are removed during the dryingprocess. This helps to maintain the protein conformation, minimizeprotein degradation during the lyophilization cycle and improve thelong-term products. Examples include polyols or sugars such as sucroseand trehalose.

The term “pharmaceutical formulation” refers to preparations which arein such form as to permit the active ingredients to be effective, andwhich contains no additional components which are toxic to the subjectsto which the formulation would be administered.

“Pharmaceutically acceptable” excipients (vehicles, additives) are thosewhich can reasonably be administered to a subject mammal to provide aneffective dose of the active ingredient employed.

“Reconstitution time” is the time that is required to rehydrate alyophilized formulation with a solution to a particle-free clarifiedsolution.

A “stable” formulation is one in which the protein therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon storage.

The term “epitope” as used herein refers to the portion of an antigenwhich is recognized by an antibody. Epitopes include, but are notlimited to, a short amino acid sequence or peptide (optionallyglycosylated or otherwise modified) enabling a specific interaction witha protein (e.g., an antibody) or ligand. For example, an epitope may bea part of a molecule to which the antigen-binding site of an antibodyattaches.

The term “treating” or “treatment” covers the treatment of a disease ordisorder (e.g., cancer), in a subject, such as a human, and includes:(i) inhibiting a disease or disorder, i.e., arresting its development;(ii) relieving a disease or disorder, i.e., causing regression of thedisorder; (iii) slowing progression of the disorder; and/or (iv)inhibiting, relieving, or slowing progression of one or more symptoms ofthe disease or disorder. In some embodiments “treating” or “treatment”refers to the killing of cancer cells.

The term “kill” with respect to a cancer treatment is directed toinclude any type of manipulation that will lead to the death of thatcancer cell or at least of portion of a population of cancer cells.

Additionally, some terms used in this specification are morespecifically defined below.

Overview

The current invention is predicated, in part, on the surprisingdiscovery that optionally lyophilized nanoparticles comprising a carrierprotein, an antibody, and a therapeutic agent provide targeted therapyto a tumor while minimizing toxicity to the patient. The nanoparticlesas described herein are thus a significant improvement versusconventional ADCs.

For conventional ADCs to be effective, it is critical that the linker bestable enough not to dissociate in the systemic circulation but allowfor sufficient drug release at the tumor site. Alley, S. C., et al.(2008) Bioconjug Chem 19:759-765. This has proven to be a major hurdlein developing effective drug conjugate (Julien, D. C., et al. (2011)MAbs 3:467-478; Alley, S. C., et al. (2008) Bioconjug Chem 19:759-765);therefore, an attractive feature of the nano-immune conjugate is that abiochemical linker is not required.

Another shortcoming of current ADCs is that higher drug penetration intothe tumor has not been substantively proven in human tumors. Earlytesting of ADCs in mouse models suggested that tumor targeting withantibodies would result in a higher concentration of the active agent inthe tumor (Deguchi, T. et al. (1986) Cancer Res 46: 3751-3755); however,this has not correlated in the treatment of human disease, likelybecause human tumors are much more heterogeneous in permeability thanmouse tumors. Jain, R. K. et al. (2010) Nat Rev Clin Oncol 7:653-664.Also, the size of the nanoparticle is critical for extravasation fromthe vasculature into the tumor. In a mouse study using a human colonadenocarcinoma xenotransplant model, the vascular pores were permeableto liposomes up to 400 nm. Yuan, F., et al. (1995) Cancer Res 55:3752-3756. Another study of tumor pore size and permeabilitydemonstrated that both characteristics were dependent on tumor locationand growth status, with regressing tumors and cranial tumors permeableto particles less than 200 nm. Hobbs, S. K., et al. (1998) Proc NatlAcad Sci USA 95:4607-4612. The nano-immune conjugate described hereinovercomes this issue by the fact that the large complex, which is lessthan 200 nm intact, is partially dissociated in systemic circulationinto smaller functional units that are easily able to permeate tumortissue. Furthermore, once the conjugate arrives to the tumor site, thesmaller toxic payload can be released and only the toxic portion needsto be taken up by tumor cells, not the entire conjugate.

The advent of antibody—(i.e. AVASTIN®) coated albumin nanoparticlescontaining a therapeutic agent (i.e., ABRAXANE®) has led to a newparadigm of directional delivery of two or more therapeutic agents to apredetermined site in vivo. See PCT Patent Publication Nos. WO2012/154861 and WO 2014/055415, each of which is incorporated herein byreference in its entirety.

When compositions of albumin and an antibody are admixed together in anaqueous solution at specific concentrations and ratios, the antibodiesspontaneously self-assemble into and onto the albumin to formnanoparticles having multiple copies of the antibody (up to 500 ormore). Without being limited to any theory, it is contemplated that theantigen receptor portion of the antibody is positioned outward from thenanoparticle while the hydrophobic tail in integrated into the albuminby hydrophobic-hydrophobic interactions.

While protein compositions comprising a single source protein arecommonly stored in lyophilized form where they exhibit significantshelf-life, such lyophilized compositions do not contain aself-assembled nanoparticle of two different proteins integratedtogether by hydrophobic-hydrophobic interactions. Moreover, thenanoparticle configuration wherein a majority of the antibody bindingportions are exposed on the surface of the nanoparticles lends itself tobeing susceptible to dislodgement or reconfiguration by conditions whichotherwise would be considered benign. For example, duringlyophilization, ionic charges on the proteins are dehydrated therebyexposing the underlying charges. Exposed charges allow for charge-chargeinteractions between the two proteins which can alter the bindingaffinity of each protein to the other. In addition, the concentration ofthe nanoparticles increases significantly as the solvent (e.g., water)is removed. Such increased concentrations of nanoparticles could lead toirreversible oligomerization. Oligomerization is a known property ofproteins that reduces the biological properties of the oligomer ascompared to the monomeric form and increases the size of the particlesometimes beyond 1 micron.

On the other hand, a stable form of a nanoparticle composition isrequired for clinical and/or commercial use where a shelf-life of atleast 3 months is required and shelf-lives of greater than 6 months or 9months are preferred. Such a stable composition must be readilyavailable for intravenous injection, must retain its self-assembled formupon intravenous injection so as to direct the nanoparticle to thepredetermined site in vivo, must have a maximum size of less than 1micron so as to avoid any ischemic event when delivered into the bloodstream, and finally must be compatible with the aqueous composition usedfor injection.

Compounds

As will be apparent to the skilled artisan upon reading this disclosure,the present disclosure relates to compositions of nanoparticlescontaining a carrier protein, antibodies, and optionally at least onetherapeutic agent, wherein said compositions are optionally lyophilized.

In some embodiments, the carrier protein can be albumin, gelatin,elastin (including topoelastin) or elastin-derived polypeptides (e.g.,α-elastin and elastin-like polypeptides (ELPs)), gliadin, legumin, zein,soy protein (e.g., soy protein isolate (SPI)), milk protein (e.g.,3-lactoglobulin (BLG) and casein), or whey protein (e.g., whey proteinconcentrates (WPC) and whey protein isolates (WPI)). In preferredembodiments, the carrier protein is albumin. In preferred embodiments,the albumin is egg white (ovalbumin), bovine serum albumin (BSA), or thelike. In even more preferred embodiments, the carrier protein is humanserum albumin (HSA). In some embodiments, the carrier protein is agenerally regarded as safe (GRAS) excipient approved by the UnitedStates Food and Drug Administration (FDA).

In some embodiments, the antibodies are selected from the groupconsisting of ado-trastuzumab emtansine, alemtuzumab, bevacizumab,cetuximab, denosumab, dinutuximab, ipilimumab, nivolumab, obinutuzumab,ofatumumab, panitumumab, pembrolizumab, pertuzumab, rituximab, andtrastuzumab. In some embodiments, the antibodies are a substantiallysingle layer of antibodies on all or part of the surface of thenanoparticle.

Table 1 depicts a list of non-limiting list of antibodies.

TABLE 1 Antibodies Antibodies Biologic Treatment(s)/Target(s) Mono-Rituximab (Rituxan ®) Non-Hodgkin lymphoma clonal Alemtuzumab(Campath ®) Chronic lymphocytic leukemia anti- (CLL) bodies Ipilimumab(Yervoy ®) Metastatic melanoma (MAbs) Bevacizumab (Avastin ®) Coloncancer, lung cancer, renal cancer, ovanan cancer, glioblastomamultiforme Cetuximab (Erbitux ®) Colorectal cancer, non-small cell lungcancer, head and neck cancer, cervical cancer, glioblastoma, ovarianepithelia, fallopian tube or primary peritoneal cancer, renal cellcancer Panitumumab (Vectibix ®) Colorectal cancer Trastuzumab(Herceptin ®) Breast cancer, Adenocarcinoma ⁹⁰Y-ibritumomab tiuxetanNon-Hodgkin lymphoma (Zevalin ®) Ado-trastuzumab emtansine Breast cancer(Kadycla ®, also called TDM- Brentuximab vedotin Hodgkin lymphoma,Anaplastic (Adcetris ®) large cell lymphoma Blinatumomab (Blincyto)Acute lymphocytic leukemia (ALL) Pembrolizumab (Keytruda ®) PD-1(melanoma, non-small cell lung cancer) Nivolumab (Opdivo ®) PD-1(melanoma, non-small cell lung cancer) Ofatumumab (Arzerra ®) Chroniclymphocytic leukemia (CLL) Pertuzumab (Perieta ®) Breast cancerObinutuzumab (Gazyva ®) Lymphoma Dinutuximab (Unituxjn ®) NeuroblastomaDenosurnab (Prolia ®) Bone metastases, multiple myeloma, giant celltumor of bone

In some embodiments, the at least one therapeutic agent is selected fromthe group consisting of abiraterone, bendamustine, bortezomib,carboplatin, cabazitaxel, cisplatin, chlorambucil, dasatinib, docetaxel,doxorubicin, epirubicin, erlotinib, etoposide, everolimus, gefitinib,idarubicin, imatinib, hydroxyurea, imatinib, lapatinib, leuprorelin,melphalan, methotrexate, mitoxantrone, nedaplatin, nilotinib,oxaliplatin, paclitaxel, pazopanib, pemetrexed, picoplatin, romidepsin,satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin,vinblastine, vinorelbine, vincristine, and cyclophosphamide.

Table 2 depicts a list of non-limiting list of cancer therapeuticagents.

TABLE 2 Cancer therapeutic agents Cancer Drus Drug Target(s) Abitrexate(Methotrexate) Acute lymphoblastic leukemia; breast cancer; gestationaltrophoblastic disease, head and neck cancer; lung cancer; mycosisfungoides; non-Hodgkin lymphoma; Abraxane (Paclitaxel Albumin-stabilizedBreast cancer; non-small cell lung cancer; Nanoparticle Formulation)pancreatic cancer ABVD (Adriamycin, bleomycin, vinblastine Hodgkinlymphoma sulfate, dacarbazine) ABVE (Adriamycin, bleomycin, vincristineHodgkin lymphoma (in children) sulfate, etoposide) ABVE-PC(Adriamycin,bleomycin, vincristine Hodgkin lymphoma (in children) sulfate,etoposide, prednisone, cyclophosphamide) AC (Adriamycincyclophosphamide) Breast cancer AC-T (Adriamycin, cylclophosphamide,Taxol) Breast cancer Adcetris (Brentuximab Vedotin) Anaplastic largecell lymphoma; Hodgkin lymphoma ADE (Cytarabine (Ara-C), DaunorubicinAcute myeloid leukemia (in children) Hydrochloride, Etoposide)Ado-Trastuzumab Emtansine Breast cancer Adriamycin (DoxorubicinHydrochloride) Acute lymphoblastic leukemia; acute myeloid leukemia;breast cancer, gastric (stomach) cancer; Hodgkin lymphoma;neuroblastoma; non-Hodgkin lymphoma; ovarian cancer; small cell lungcancer; soft tissue and bone sarcomas; thyroid cancer; transitional cellbladder cancer; Wilms tumor Adrucil (Fluorouracil) Basal cell carcinoma;breast cancer; colorectal cancer; gastric (stomach) adenocarcinoma;pancreatic cancer; squamous the head and neck Afatinib DimaleateNon-small cell lung cancer Afinitor (Everolimus) Breast cancer,pancreatic cancer; renal cell carcinoma; subependymal giant cellastrocytoma Alimta (Pemetrexed Disodium) Malignant pleural mesothelioma;non-small cell lung cancer Ambochlorin (Chlorambucil) Chroniclymphocytic leukemia; Hodgkin lymphoma; non-Hodgkin Anastrozole Breastcancer Aredia (Pamidronate Disodium) Breast cancer; multiple myelomaArimidex (Anastrozole) Breast cancer Aromasin (Exemestane) Advancedbreast cancer; early-stage breast cancer and estrogen receptor Arranon(Nelarabine) T-cell acute lymphoblastic leukemia; T-cell lymphoblasticlymphoma Azacitidine Myelodysplastic syndromes BEACOPP Hodgkin lymphomaBecenum (Carmustine) Brain tumors; Hodgkin lymphoma; multiple myeloma;non-Hodgkin Beleodaq (Belinostat) Peripheral T-cell lymphoma BEP Ovariangerm cell tumors; testicular germ cell tumors Bicalutamide Prostatecancer BiCNU (Carmustine) Brain tumors; Hodgkin lymphoma; multiplemyeloma; non-Hodgkin Bleomycin Hodgkin lymphoma; non-Hodgkin lymphoma;penile cancer; squamous cell carcinoma of the cervix; squamous cellcarcinoma of the head and neck; squamous cell carcinoma of the vulva;testicular cancer Bosulif (Bosutinib) Chronic myelogenous leukemiaBrentuximab Vedotin Anaplastic large cell lymphoma; Hodgkin lymphomaBusulfan Chronic myelogenous leukemia Busulfex (Busulfan) Chronicmyelogenous leukemia Cabozantinib-S-Malate Medullary thyroid cancer CAFBreast cancer Camptosar (Irinotecan Hydrochloride) Colorectal cancerCAPOX Colorectal cancer Carfilzomib Multiple myeloma Casodex(Bicalutamide) Prostate cancer CeeNU (Lomustine) Brain tumors; Hodgkinlymphoma Ceritinib Non-small cell lung cancer Cerubidine (DaunorubicinHydrochloride) Acute lymphoblastic leukemia; acute myeloid leukemiaChlorambucil Chronic lymphocytic leukemia; Hodgkin lymphoma; non-HodgkinCHLORAMBUCIL-PREDNISONE Chronic lymphocytic leukemia CHOP Non-Hodgkinlymphoma Cisplatin Bladder cancer; cervical cancer; malignantmesothelioma; non-small cell lung cancer; ovarian cancer; squamous cellcarcinoma of the head and neck; testicular cancer Clafen(Cyclophosphamide) Acute lymphoblastic leukemia; acute myeloid leukemia;breast cancer; chronic lymphocytic leukemia; chronic myelogenousleukemia; Hodgkin lymphoma; multiple myeloma; mycosis fungoides;neuroblastoma; non- Hodgkin lymphoma; ovarian cancer; retinoblastomaClofarex (Clofarabine) Acute lymphoblastic leukemia CMF Breast cancerCometriq (Cabozantinib-S-Malate) Medullary thyroid cancer COPP Hodgkinlymphoma; non-Hodgkin lymphoma COPP-ABV Hodgkin lymphoma Cosmegen(Dactinomycin) Ewing sarcoma; gestational trophoblastic disease;rhabdomyosarcoma; solid tumors; testicular cancer; Wilms tumor CVPNon-Hodgkin lymphoma; chronic lymphocytic leukemia CyclophosphamideAcute lymphoblastic leukemia; acute myeloid leukemia; breast cancer;chronic lymphocytic leukemia; chronic myelogenous leukemia; Hodgkinlymphoma; multiple myeloma; mycosis fungoides; neuroblastoma; non-Hodgkin lymphoma; ovarian cancer; retinoblastoma. Cyfos (Ifosfamide)Testicular germ cell tumors Cyramza (Ramucirumab) Adenocarcinoma;colorectal cancer; non-small cell lung cancer Cytarabine Acutelymphoblastic leukemia; acute myeloid leukemia; chronic myelogenousleukemia; meningeal leukemia Cytosar-U (Cytarabine) Acute lymphoblasticleukemia; acute myeloid leukemia; chronic myelogenous leukemia;meningeal leukemia Cytoxan (Cyclophosphamide) Acute lymphoblasticleukemia; acute myeloid leukemia; breast cancer; chronic lymphocyticleukemia; chronic myelogenous Hodgkin lymphoma; multiple myeloma;mycosis fungoides; neuroblastoma; non- Hodgkin lymphoma; ovarian cancer;Dacarbazine Hodgkin lymphoma; melanoma Dacogen (Decitabine)Myelodysplastic syndromes Dactinomycin Ewing sarcoma; gestationaltrophoblastic disease; rhabdomyosarcoma; solid tumors; testicularcancer; Wilms tumor Daunorubicin Hydrochloride Acute lymphoblasticleukemia; acute myeloid leukemia Degarelix Prostate cancer DenileukinDiftitox Cutaneous T-cell lymphoma Denosumab Giant cell tumor of thebone; breast cancer, prostate cancer DepoCyt (Liposomal Cytarabine)Lymphomatous meningitis DepoFoam (Liposomal Cytarabine) Lymphomatousmeningitis Docetaxel Breast cancer; adenocarcinoma of the stomach orgastroesophageal junction; non-small cell lung cancer; prostate cancer;squamous cell carcinoma of the head and Doxil (Doxorubicin HydrochlorideLiposome) AIDS-related Kaposi sarcoma; multiple myeloma; ovarian cancerDoxorubicin Hydrochloride Acute lymphoblastic leukemia; acute myeloidleukemia; breast cancer; gastric (stomach) cancer; Hodgkin lymphoma;neuroblastoma; non-Hodgkin lymphoma; ovarian cancer; small cell lungcancer; soft tissue and bone sarcomas; thyroid cancer; transitional cellbladder cancer; Wilms tumor. Dox-SL (Doxorubicin HydrochlorideAIDS-related Kaposi sarcoma; multiple Liposome) myeloma; ovarian cancerDTIC-Dome (Dacarbazine) Hodgkin lymphoma; melanoma Efudex (Fluorouracil)Basal cell carcinoma; breast cancer; colorectal cancer; gastric(stomach) adenocarcinoma; pancreatic cancer; squamous cell carcinoma ofthe head and neck Ellence (Epirubicin Hydrochloride) Breast cancerEloxatin (Oxaliplatin) Colorectal cancer; stage III colon cancer Emend(Aprepitant) Nausea and vomiting caused by chemotherapy and nausea andvomiting after Enzalutamide Prostate cancer Epirubicin HydrochlorideBreast cancer EPOCH Non-Hodgkin lymphoma Erbitux (Cetuximab) Colorectalcancer; squamous cell carcinoma of the head and neck Eribulin MesylateBreast cancer Erivedge (Vismodegib) Basal cell carcinoma ErlotinibHydrochloride Non-small cell lung cancer; pancreatic cancer Erwinaze(Asparaginase Acute lymphoblastic leukemia Erwinia chrysanthemi)Etopophos (Etoposide Phosphate) Small cell lung cancer; testicularcancer Evacet (Doxorubicin Hydrochloride Liposome) AIDS-related Kaposisarcoma; multiple myeloma; ovarian cancer Everolimus Breast cancer;pancreatic cancer; renal cell carcinoma; subependymal giant cellastrocytoma Evista (Raloxifene Hydrochloride) Breast cancer ExemestaneBreast cancer Fareston (Toremifene) Breast cancer Farydak (Panobinostat)Multiple myeloma Faslodex (Fulvestrant) Breast cancer FEC Breast cancerFemara (Letrozole) Breast cancer Filgrastim Neutropenia Fludara(Fludarabine Phosphate) Chronic lymphocytic leukemia Fluoroplex(Fluorouracil) Basal cell carcinoma; breast cancer; colorectal cancer;gastric (stomach) adenocarcinoma; pancreatic cancer; squamous cellcarcinoma of the head and neck Folex (Methotrexate) Acute lymphoblasticleukemia; breast cancer; gestational trophoblastic disease; head andneck cancer; lung cancer; mycosis fungoides; non-Hodgkin lymphoma;FOLFIRI Colorectal cancer FOLFIRI-BEVACIZUMAB Colorectal cancerFOLFIRI-CETUXIMAB Colorectal cancer FOLFIRINOX Pancreatic cancer FOLFOXColorectal cancer Folotyn (Pralatrexate) Peripheral T-cell lymphomaFU-LV Colorectal cancer; esophageal cancer; gastric cancer FulvestrantBreast cancer Gefitinib Non-small cell lung cancer GemcitabineHydrochloride Breast cancer; non-small cell lung cancer; ovarian cancer;pancreatic cancer GEMCITABINE-CISPLATIN Biliary tract cancer; bladdercancer; cervical cancer; malignant mesothelioma; non-small cell lungcancer; ovarian cancer; pancreatic cancer GEMCITABINE-OXALIPLATINPancreatic cancer Gemtuzumab Ozogamicin (antibody drug Acute myeloidleukemia conjugate) Gemzar (Gemcitabine Hydrochloride) Breast cancer;non-small cell lung cancer; ovarian cancer; pancreatic cancer Gilotrif(Afatinib Dimaleate) Non-small cell lung cancer Gleevec (ImatinibMesylate) Acute lymphoblastic leukemia; chronic eosinophilic leukemia orhypereosinophilic syndrome; chronic myelogenous leukemia;dermatofibrosarcoma protuberans; gastrointestinal stromal tumor;myelodysplastic/myeloproliferative neoplasms; systemic mastocytosis.Gliadel (Carmustine Implant) Glioblastoma multiforme; malignant gliomaGoserelin Acetate Breast cancer; prostate cancer Halaven (EribulinMesylate) Breast cancer Hycamtin (Topotecan Hydrochloride) Cervicalcancer; ovarian cancer; small cell lung cancer Hyper-CVAD Acutelymphoblastic leukemia; non-Hodgkin lymphoma Ibrance (Palbociclib)Breast cancer Ibrutinib Chronic lymphocytic leukemia; mantel celllymphoma; ICE Hodgkin lymphoma; non-Hodgkin lymphoma Iclusig (PonatinibHydrochloride) Acute lymphoblastic leukemia; Chronic myelogenousleukemia Idamycin (Idarubicin Hydrochloride) Acute myeloid leukemiaImatinib Mesylate Acute lymphoblastic leukemia; chronic eosinophilicleukemia or hypereosinophilic syndrome; chronic myelogenous leukemia;dermatofibrosarcoma protuberans; gastrointestinal stromal tumor;myelodysplastic/myeloproliferative neoplasms; systemic mastocytosis.Imbruvica (Ibrutinib) Chronic lymphocytic leukemia; mantle celllymphoma; Waldenstr6m Inlyta (Axitinib) Renal cell carcinoma Iressa(Gefitinib) Non-small cell lung cancer Irinotecan HydrochlorideColorectal cancer Istodax (Romidepsin) Cutaneous T-cell lymphoma Ixempra(Ixabepilone) Breast cancer Jevtana (Cabazitaxel) Prostate cancerKeoxifene (Raloxifene Hydrochloride) Breast cancer Kyprolis(Carfilzomib) Multiple myeloma Lenvima (Lenvatinib Mesylate) Thyroidcancer Letrozole Breast cancer Leucovorin Calcium Colorectal cancerLeukeran (Chlorambucil) Chronic lymphocytic leukemia; Hodgkin lymphoma;non-Hodgkin Leuprolide Acetate Prostate cancer Linfolizin (Chlorambucil)Chronic lymphocytic leukemia; Hodgkin lymphoma; non-Hodgkin LipoDox(Doxorubicin Hydrochloride AIDS-related Kaposi sarcoma; multipleLiposome) myeloma; ovarian cancer Lomustine Brain tumors; Hodgkinlymphoma Lupron (Leuprolide Acetate) Prostate cancer Lynparza (Olaparib)Ovarian cancer Marqibo (Vincristine Sulfate Liposome) Acutelymphoblastic leukemia Matulane (Procarbazine Hydrochloride) Hodgkinlymphoma Mechlorethamine Hydrochloride Bronchogenic carcinoma; chroniclymphocytic leukemia; chronic myelogenous leukemia; Hodgkin lymphoma;malignant pleural effusion, malignant pericardial effusion, andmalignant peritoneal effusion; mycosis fungoides; non-Hodgkin lymphomaMegace (Megestrol Acetate) Breast cancer; endometrial cancer Mekinist(Trametinib) Melanoma Mercaptopurine Acute lymphoblastic leukemia Mesnex(Mesna) Hemorrhagic cystitis Methazolastone (Temozolomide) Anaplasticastrocytoma; glioblastoma multiforme Mexate (Methotrexate) Acutelymphoblastic leukemia; breast cancer; gestational trophoblasticdisease; head and neck cancer; lung cancer; mycosis fungoides;non-Hodgkin lymphoma; Mexate-AQ (Methotrexate) Acute lymphoblasticleukemia; breast cancer; gestational trophoblastic disease; head andneck cancer; lung cancer; mycosis fungoides; non-Hodgkin lymphoma;Mitoxantrone Hydrochloride Acute myeloid leukemia; prostate cancerMitozytrex (Mitomycin C) Gastric (stomach) and pancreatic adenocarcinomaMOPP Hodgkin lymphoma Mozobil (Plerixafor) Multiple myeloma; non-Hodgkinlymphoma Mustargen (Mechlorethamine Hydrochloride) Bronchogeniccarcinoma; chronic lymphocytic leukemia; chronic myelogenous leukemia;Hodgkin lymphoma; malignant pleural effusion, malignant pericardialeffusion, and malignant peritoneal effusion; mycosis fungoides;non-Hodgkin lymphoma Myleran (Busulfan) Chronic myelogenous leukemiaMylotarg (Gemtuzumab Ozogamicin) Acute myeloid leukemia NanoparticlePaclitaxel (Paclitaxel Albumin- Breast cancer; Non-small cell lungcancer; stabilized Nanoparticle Formulation) Pancreatic cancer Navelbine(Vinorelbine Tartrate) Non-small cell lung cancer Nelarabine T-cellacute lymphoblastic leukemia Neosar (Cyclophosphamide) Acutelymphoblastic leukemia; Acute myeloid leukemia; Breast cancer; Chroniclymphocytic leukemia; Chronic myelogenous leukemia; Hodgkin lymphoma;Multiple myeloma; Mycosis fungoides; Neuroblastoma; Non- Hodgkinlymphoma; Ovarian cancer; Retinoblastoma Nexavar (Sorafenib Tosylate)Hepatocellular carcinoma; Renal cell carcinoma; Thyroid cancer NilotinibChronic myelogenous leukemia Nivolumab Melanoma; Squamous non-small celllung cancer Nolvadex (Tamoxifen Citrate) Breast cancer Odomzo(Sonidegib) Basal cell carcinoma OEPA Hodgkin lymphoma OFF Pancreaticcancer Olaparib Ovarian cancer Oncaspar (Pegaspargase) Acutelymphoblastic leukemia OPPA Hodgkin lymphoma Oxaliplatin Colorectalcancer; Stage III colon cancer Paclitaxel AIDS-related Kaposi sarcoma;Breast cancer; Non-small cell lung cancer; Ovarian PaclitaxelAlbumin-stabilized Nanoparticle Breast cancer; Non-small lung cancer;Formulation Pancreatic cancer PAD Multiple myeloma Palbociclib Breastcancer Pamidronate Disodium Breast cancer; Multiple myeloma PanitumumabColorectal cancer Panobinostat Multiple myeloma Paraplat (Carboplatin)Non-small cell lung cancer; Ovarian cancer Paraplatin (Carboplatin)Non-small cell lung cancer; Ovarian cancer Pazopanib Hydrochloride Renalcell carcinoma; Soft tissue sarcoma Pegaspargase Acute lymphoblasticleukemia Pemetrexed Disodium Malignant pleural mesothelioma; Non-smallcell lung cancer Platinol (Cisplatin) Bladder cancer; Cervical cancer;Malignant mesothelioma; Non-small cell lung cancer; Ovarian cancer;Squamous cell carcinoma of the head and neck; Testicular cancerPlatinal-AQ (Cisplatin) Bladder cancer; Cervical cancer; Malignantmesothelioma; Non-small cell lung cancer; Ovarian cancer; Squamous cellcarcinoma of the head and neck; Testicular cancer Plerixafor Multiplemyeloma; Non-Hodgkin lymphoma Pomalidomide Multiple myeloma Pomalyst(Pomalidomide) Multiple myeloma Pontinib Hydrochloride Acutelymphoblastic leukemia; Chronic myelogenous leukemia PralatrexatePeripheral T-cell lymphoma Prednisone Acute lymphoblastic leukemia;Chronic lymphocytic leukemia; Hodgkin lymphoma; Multiple myeloma;Non-Hodgkin lymphoma; Prostate cancer; Thymoma and thymic carcinomaProcarbazine Hydrochloride Hodgkin lymphoma Provenge (Sipuleucel-T)Prostate cancer Purinethol (Mercaptopurine) Acute lymphoblastic leukemiaRadium 223 Dichloride Prostate cancer Raloxifene Hydrochloride Breastcancer R-CHOP Non-Hodgkin lymphoma R-CVP Non-Hodgkin lymphomaRegorafenib Colorectal cancer; Gastrointestinal stromal tumor R-EPOCHB-cell non-Hodgkin lymphoma Revlimid (Lenalidomide) Mantle celllymphoma; Multiple myeloma; Anemia Rheumatrex (Methotrexate) Acutelymphoblastic leukemia; Breast cancer; Gestational trophoblasticdisease; Head and neck cancer; Lung cancer; Non-Hodgkin lymphoma;Osteosarcoma Romidepsin Cutaneous T-cell lymphoma Rubidomycin(Daunorubicin Hydrochloride) Acute lymphoblastic leukemia; Acute myeloidleukemia Sipuleucel-T Prostate cancer Somatuline Depot (LanreotideAcetate) Gastroenteropancreatic neuroendocrine tumors Sonidegib Basalcell carcinoma Sorafenib Tosylate Hepatocellular carcinoma; Renal cellcarcinoma; Thyroid cancer Sprycel (Dasatinib) Acute lymphoblasticleukemia; Chronic myelogenous leukemia STANFORD V Hodgkin lymphomaStivarga (Regorafenib) Colorectal cancer; Gastrointestinal stromal tumorSunitnib Malate Gastronintestinal stromal tumor; Pancreatic cancer;Renal cell carcinoma Sutent (Sunitinib Malate) Gastronintestinal stromaltumor; Pancreatic cancer; Renal cell carcinoma Synovir (Thalidomide)Multiple myeloma Synribo (Omacetaxine Mepesuccinate) Chronic myelogenousleukemia TAC Breast cancer Tafinlar (Dabrafenib) Melanoma TamoxifenCitrate Breast cancer Tarabine PFS (Cytarabine) Acute lymphoblasticleukemia; Acute myeloid leukemia; Chronic myelogenous Tarceva (ErlotinibHydrochloride) Non-small cell lung cancer; Pancreatic cancer Targretin(Bexarotene) Skin problems caused by cutaneous T-cell lymphoma Tasigna(Niltinib) Chronic myelogenous leukemia Taxol (Paclitaxel) AIDS-relatedKaposi sarcoma; Breast cancer; Non-small cell lung cancer; OvarianTaxotere (Docetaxel) Breast cancer; Adenocarcinoma; Non-small cell lungcancer; Prostate cancer; Squamous cell carcinoma of the head and Temodar(Temozolomide) Anaplastic astrocytoma; Glioblastoma multiformeTemozolomide Anaplastic astrocytoma; Glioblastoma multiforme ThiotepaBladder cancer; Breast cancer; Malignant pleural effusion, malignantpericardial effusion, and malignant peritoneal effusion; Ovarian cancerToposar (Etoposide) Small cell lung cancer; Testicular cancer TopotecanHydrochloride Cervical cancer; Ovarian cancer; Small cell lung cancerToremifene Breast cancer Torisel (Temsirolimus) Renal cell carcinoma TPFSquamous cell carcinoma of the head and neck; Gastric (stomach) cancerTrastuzumab Adenocarcinoma; Breast cancer Treanda (BendamustineHydrochloride) B-cell non-Hodgkin lymphoma; Chronic lymphocytic leukemiaTrisenox (Arsenic Trioxide) Acute promyelocytic leukemia Tykerb(Lapatinib Ditosylate) Breast cancer Vandetabib Medullary thyroid cancerVAMP Hodgkin lymphoma VeIP Ovarian germ cell; Testicular cancer Velban(Vinblastine Sulfate) Breast cancer; Choriocarcinoma; Hodgkin lymphoma;Kaposi sarcoma; Mycosid fungoides; Non-Hodgkin Testicular cancer Velcade(Bortezomib) Mulitple myeloma; Mantle cell lymphoma Velsar (VinblastineSulfate) Breast cancer; Choriocarcinoma; Hodgkin lymphoma; Kaposisarcoma; Mycosis fungoides; Non-Hodgkin lymphoma; Testicular cancerVePesid (Etoposide) Small cell lung cancer; Testicular cancer Viadur(Leuprolide Acetate) Prostate cancer Vidaza (Azacitidine)Myelodysplastic syndromes Vincasar PFS (Vincristine Sulfate) Acuteleukemia; Hodgkin lymphoma; Neuroblastoma; Non-Hodgkin lymphoma;Rhabdomyosarcoma; Wilms Vincristine Sulfate Liposome Acute lymphoblasticleukemia Vinorelbine Tartrate Non-small cell lung cancer VIP Testicularcancer Visbodegib Basal cell carcinoma Voraxaze (Glucarpidase) Toxicblood levels of the anticancer drug methotrexate Votrient (PazopanibHydrochloride) Renal cell carcinoma; Soft tissue sarcoma Wellcovorin(Leucovorin Calcium) Colorectal cancer; Anemia Xalkori (Crizotinib)Non-small cell lung cancer Xeloda (Capecitabine) Breast cancer;Colorectal cancer XELIRI Colorectal cancer; Esophageal cancer; Gastric(stomach) cancer XELOX Colorectal cancer Xofigo (Radium 223 Dichloride)Prostate cancer Xtandi (Enzalutamide) Prostate cancer Zaltrap(Ziv-Aflibercept) Colorectal cancer Zelboraf (Vemurafenib) MelanomaZiv-Aflibercept Colorectal cancer Zoladex (Goserelin Acetate) Breastcancer; Prostate cancer Zolinza (Vorinostat) Cutaneous T-cell lymphomaZometa (Zoledronic Acid) Multiple myeloma Zydelig (Idelalisib) Chroniclymphocytic leukemia; Non-Hodgkin lymphoma (Follicula B-cell non Hodgkinlymphoma and Small lymphocytic Zykadia (Certinib) Non-small cell lungcancer Zytiga (Abiraterone Acetate) Prostate cancer

It is to be understood that the therapeutic agent may be located insidethe nanoparticle, on the outside surface of the nanoparticle, or both.The nanoparticle may contain more than one therapeutic agent, forexample, two therapeutic agents, three therapeutic agents, fourtherapeutic agents, five therapeutic agents, or more. Furthermore, ananoparticle may contain the same or different therapeutic agents insideand outside the nanoparticle.

In some embodiments any carrier protein, antibody, therapeutic agent, orany combination thereof is expressly excluded. For example in someembodiments a composition may comprise any carrier protein andchemotherapeutic except Abraxane® and/or any targeting antibody exceptbevacizumab.

In one aspect, the nanoparticle comprises at least 100 antibodiesnon-covalently bound to the surface of the nanoparticle. In one aspect,the nanoparticle comprises at least 200 antibodies non-covalently boundto the surface of the nanoparticle. In one aspect, the nanoparticlecomprises at least 300 antibodies non-covalently bound to the surface ofthe nanoparticle. In one aspect, the nanoparticle comprises at least 400antibodies non-covalently bound to the surface of the nanoparticle. Inone aspect, the nanoparticle comprises at least 500 antibodiesnon-covalently bound to the surface of the nanoparticle. In one aspect,the nanoparticle comprises at least 600 antibodies non-covalently boundto the surface of the nanoparticle.

In one aspect, the nanoparticle comprises between about 100 and about1000 antibodies non-covalently bound to the surface of the nanoparticle.In one aspect, the nanoparticle comprises between about 200 and about1000 antibodies non-covalently bound to the surface of the nanoparticle.In one aspect, the nanoparticle comprises between about 300 and about1000 antibodies non-covalently bound to the surface of the nanoparticle.In one aspect, the nanoparticle comprises between about 400 and about1000 antibodies non-covalently bound to the surface of the nanoparticle.In one aspect, the nanoparticle comprises between about 500 and about1000 antibodies non-covalently bound to the surface of the nanoparticle.In one aspect, the nanoparticle comprises between about 600 and about1000 antibodies non-covalently bound to the surface of the nanoparticle.In one aspect, the nanoparticle comprises between about 200 and about800 antibodies non-covalently bound to the surface of the nanoparticle.In one aspect, the nanoparticle comprises between about 300 and about800 antibodies non-covalently bound to the surface of the nanoparticle.In preferred embodiments, the nanoparticle comprises between about 400and about 800 antibodies non-covalently bound to the surface of thenanoparticle. Contemplated values include any value or subrange withinany of the recited ranges, including endpoints.

In one aspect, the average particle size in the nanoparticle compositionis less than about 1 μm. In one aspect, the average particle size in thenanoparticle composition is between about 130 nm and about 1 μm. In oneaspect, the average particle size in the nanoparticle composition isbetween about 130 nm and about 900 nm. In one aspect, the averageparticle size in the nanoparticle composition is between about 130 nmand about 800 nm. In one aspect, the average particle size in thenanoparticle composition is between about 130 nm and about 700 nm. Inone aspect, the average particle size in the nanoparticle composition isbetween about 130 nm and about 600 nm. In one aspect, the averageparticle size in the nanoparticle composition is between about 130 nmand about 500 nm. In one aspect, the average particle size in thenanoparticle composition is between about 130 nm and about 400 nm. Inone aspect, the average particle size in the nanoparticle composition isbetween about 130 nm and about 300 nm. In one aspect, the averageparticle size in the nanoparticle composition is between about 130 nmand about 200 nm. In a preferred embodiment, the average particle sizein the nanoparticle composition is between about 150 nm and about 180nm. In an especially preferred embodiment, the mean particle size in thenanoparticle composition is about 160 nm. Contemplated values includeany value, subrange, or range within any of the recited ranges,including endpoints.

In one aspect, the nanoparticle composition is formulated forintravenous injection. In order to avoid an ischemic event, thenanoparticle composition formulated for intravenous injection shouldcomprise nanoparticles with an average particle size ofless than about 1μm.

In one aspect, the average particle size in the nanoparticle compositionis greater than about 1 μm. In one aspect, the average particle size inthe nanoparticle composition is between about 1 μm and about 5 μm. Inone aspect, the average particle size in the nanoparticle composition isbetween about 1 μm and about 4 μm. In one aspect, the average particlesize in the nanoparticle composition is between about 1 μm and about 3μm. In one aspect, the average particle size in the nanoparticlecomposition is between about 1 μm and about 2 μm. In one aspect, theaverage particle size in the nanoparticle composition is between about 1μm and about 1.5 μm. Contemplated values include any value, subrange, orrange within any of the recited ranges, including endpoints.

In one aspect, the nanoparticle composition is formulated for directinjection into a tumor. Direct injection includes injection into orproximal to a tumor site, perfusion into a tumor, and the like. Whenformulated for direct injection into a tumor, the nanoparticle maycomprise any average particle size. Without being bound by theory, it isbelieved that larger particles (e.g., greater than 500 nm, greater than1 μm, and the like) are more likely to be immobilized within the tumor,thereby providing a beneficial effect. Larger particles can accumulatein the tumor or specific organs. See, e.g., 20-60 micron glass particlethat is used to inject into the hepatic artery feeding a tumor of theliver, called “TheraSphere®” (in clinical use for liver cancer).Therefore, for intravenous administration, particles under 1 μm aretypically used. Particles over 1 μm are, more typically, administereddirectly into a tumor (“direct injection”) or into an artery feedinginto the site of the tumor.

In one aspect, less than about 0.01% of the nanoparticles within thecomposition have a particle size greater than 200 nm, greater than 300nm, greater than 400 nm, greater than 500 nm, greater than 600 nm,greater than 700 nm, or greater than 800 nm. In one aspect, less thanabout 0.001% of the nanoparticles within the composition have a particlesize greater than 200 nm, greater than 300 nm, greater than 400 nm,greater than 500 nm, greater than 600 nm, greater than 700 nm, orgreater than 800 nm. In a preferred embodiment, less than about 0.01% ofthe nanoparticles within the composition have a particle size greaterthan 800 nm. In a more preferred embodiment, less than about 0.001% ofthe nanoparticles within the composition have a particle size greaterthan 800 nm.

In a preferred aspect, the sizes and size ranges recited herein relateto particle sizes of the reconstituted lyophilized nanoparticlecomposition. That is, after the lyophilized nanoparticles areresuspended in an aqueous solution (e.g., water, other pharmaceuticallyacceptable excipient, buffer, etc.), the particle size or averageparticle size is within the range recited herein.

In one aspect, at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, 99.5%, or 99.9% of the nanoparticles are present in thereconstituted composition as single nanoparticles. That is, fewer thanabout 50%, 40%, 30%, etc. of the nanoparticles are dimerized ormultimerized (oligomerized).

In some embodiments, the size of the nanoparticle can be controlled bythe adjusting the amount (e.g., ratio) of carrier protein to antibody.The size of the nanoparticles, and the size distribution, is alsoimportant. The nanoparticles of the invention may behave differentlyaccording to their size. At large sizes, an agglomeration may blockblood vessels. Therefore, agglomeration of nanoparticles can affect theperformance and safety of the composition. On the other hand, largerparticles may be more therapeutic under certain conditions (e.g., whennot administered intravenously).

In one aspect, the nanoparticle composition comprises at least oneadditional therapeutic agent. In one embodiment, the at least oneadditional therapeutic agent is non-covalently bound to the outsidesurface of the nanoparticle. In one embodiment, the at least oneadditional therapeutic agent is arranged on the outside surface of thenanoparticle. In one embodiment, the at least one additional therapeuticagent is selected from the group consisting of abiraterone,bendamustine, bortezomib, carboplatin, cabazitaxel, cisplatin,chlorambucil, dasatinib, docetaxel, doxorubicin, epirubicin, erlotinib,etoposide, everolimus, gemcitabine, gefitinib, idarubicin, imatinib,hydroxyurea, imatinib, lapatinib, leuprorelin, melphalan, methotrexate,mitoxantrone, nedaplatin, nilotinib, oxaliplatin, pazopanib, pemetrexed,picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib,teniposide, triplatin, vinblastine, vinorelbine, vincristine, andcyclophosphamide. In one embodiment, the at least one additionaltherapeutic agent is an anti-cancer antibody.

Methods of Making Nanoparticles

In some aspects, the current invention relates to methods of makingnanoparticle compositions as described herein.

In one aspect, the nanoparticles of the nanoparticle composition areformed by contacting the carrier protein or carrier protein-therapeuticagent particle with the antibody at a ratio of about 10:1 to about 10:30carrier protein particle or carrier protein-therapeutic agent particleto antibody. In one embodiment, the ratio is about 10:2 to about 10:25.In one embodiment, the ratio is about 10:2 to about 1:1. In a preferredembodiment, the ratio is about 10:2 to about 10:6. In an especiallypreferred embodiment, the ratio is about 10:4. Contemplated ratiosinclude any value, subrange, or range within any of the recited ranges,including endpoints.

In one embodiment, the amount of solution or other liquid mediumemployed to form the nanoparticles is particularly important. Nonanoparticles are formed in an overly dilute solution of the carrierprotein (or carrier protein-therapeutic agent) and the antibodies. Anoverly concentrated solution will result in unstructured aggregates. Insome embodiments, the amount of solution (e.g., sterile water, saline,phosphate buffered saline) employed is between about 0.5 mL of solutionto about 20 mL of solution. In some embodiments, the amount of carrierprotein is between about 1 mg/mL and about 100 mg/mL. In someembodiments, the amount of antibody is between about 1 mg/mL and about30 mg/mL. For example, in some embodiments, the ratio of carrierprotein:antibody:solution is approximately 9 mg of carrier protein(e.g., albumin) to 4 mg of antibody (e.g., BEV) in 1 mL of solution(e.g., saline). An amount of therapeutic agent (e.g., taxol) can also beadded to the carrier protein. For example, 1 mg of taxol can be added 9mg of carrier protein (10 mg carrier protein-therapeutic) and 4 mg ofantibody in 1 mL of solution. When using a typical i.v. bag, forexample, with the solution of approximately 1 liter one would need touse 1000× the amount of carrier protein/carrier protein-therapeuticagent and antibodies compared to that used in 1 mL. Thus, one cannotform the present nanoparticles in a standard i.v. bag. Furthermore, whenthe components are added to a standard i.v. bag in the therapeuticamounts of the present invention, the components do not self-assemble toform nanoparticles.

In one embodiment, the carrier protein or carrier protein-therapeuticagent particle is contacted with the antibody in a solution having a pHbetween about 4 and about 8. In one embodiment, the carrier protein orcarrier protein-therapeutic agent particle is contacted with theantibody in a solution having a pH of about 4. In one embodiment, thecarrier protein or carrier protein-therapeutic agent particle iscontacted with the antibody in a solution having a pH of about 5. In oneembodiment, the carrier protein or carrier protein-therapeutic agentparticle is contacted with the antibody in a solution having a pH ofabout 6. In one embodiment, the carrier protein or carrierprotein-therapeutic agent particle is contacted with the antibody in asolution having a pH of about 7. In one embodiment, the carrier proteinor carrier protein-therapeutic agent particle is contacted with theantibody in a solution having a pH of about 8. In a preferredembodiment, the carrier protein or carrier protein-therapeutic agentparticle is contacted with the antibody in a solution having a pHbetween about 5 and about 7.

In one embodiment, the carrier protein particle or carrierprotein-therapeutic agent particle is incubated with the antibody at atemperature of about 5° C. to about 60° C., or any range, subrange, orvalue within that range including endpoints. In a preferred embodiment,the carrier protein particle or carrier protein-therapeutic agentparticle is incubated with the antibody at a temperature of about 23° C.to about 60° C.

Without being bound by theory, it is believed that the stability of thenanoparticles within the nanoparticle composition is, at least in part,dependent upon the temperature and/or pH at which the nanoparticles areformed, as well as the concentration of the components (i.e., carrierprotein, antibody, and optionally therapeutic agent) in the solution. Inone embodiment, the K_(d) of the nanoparticles is between about 1×10⁻¹¹M and about 2×10⁻⁵ M. In one embodiment, the K_(d) of the nanoparticlesis between about 1×10⁻¹¹ M and about 2×10⁻⁸ M. In one embodiment, theK_(d) of the nanoparticles is between about 1×10⁻¹¹ M and about 7×10⁻⁹M. In a preferred embodiment, the K_(d) of the nanoparticles is betweenabout 1×10⁻¹¹ M and about 3×10⁻⁸M. Contemplated values include anyvalue, subrange, or range within any of the recited ranges, includingendpoints.

Lyophilization

The lyophilized compositions of this invention are prepared by standardlyophilization techniques with or without the presence of stabilizers,buffers, etc. Surprisingly, these conditions do not alter the relativelyfragile structure of the nanoparticles. Moreover, at best, thesenanoparticles retain their size distribution upon lyophilization and,more importantly, can be reconstituted for in vivo administration (e.g.,intravenous delivery) in substantially the same form and ratios as iffreshly made.

Formulations

In one aspect, the nanoparticle composition is formulated for directinjection into a tumor. Direct injection includes injection into orproximal to a tumor site, perfusion into a tumor, and the like. Becausethe nanoparticle composition is not administered systemically, ananoparticle composition is formulated for direct injection into a tumormay comprise any average particle size. Without being bound by theory,it is believed that larger particles (e.g., greater than 500 nm, greaterthan 1 μm, and the like) are more likely to be immobilized within thetumor, thereby providing what is believed to be a better beneficialeffect.

In another aspect, provided herein is a composition comprising acompound provided herein, and at least one pharmaceutically acceptableexcipient.

In general, the compounds provided herein can be formulated foradministration to a patient by any of the accepted modes ofadministration. Various formulations and drug delivery systems areavailable in the art. See, e.g., Gennaro, A. R., ed. (1995) Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Co.

In general, compounds provided herein will be administered aspharmaceutical compositions by any one of the following routes: oral,systemic (e.g., transdermal, intranasal or by suppository), orparenteral (e.g., intramuscular, intravenous or subcutaneous)administration.

The compositions are comprised of, in general, a compound of the presentinvention in combination with at least one pharmaceutically acceptableexcipient. Acceptable excipients are non-toxic, aid administration, anddo not adversely affect the therapeutic benefit of the claimedcompounds. Such excipient may be any solid, liquid, semi-solid or, inthe case of an aerosol composition, gaseous excipient that is generallyavailable to one of skill in the art.

Solid pharmaceutical excipients include starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk and the like. Liquid and semisolid excipientsmay be selected from glycerol, propylene glycol, water, ethanol andvarious oils, including those of petroleum, animal, vegetable orsynthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesameoil, etc. Preferred liquid carriers, particularly for injectablesolutions, include water, saline, aqueous dextrose, and glycols. Othersuitable pharmaceutical excipients and their formulations are describedin Remington's Pharmaceutical Sciences, edited by E. W. Martin (MackPublishing Company, 18th ed., 1990).

The present compositions may, if desired, be presented in a pack ordispenser device containing one or more unit dosage forms containing theactive ingredient. Such a pack or device may, for example, comprisemetal or plastic foil, such as a blister pack, or glass, and rubberstoppers such as in vials. The pack or dispenser device may beaccompanied by instructions for administration. Compositions comprisinga compound of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabeled for treatment of an indicated condition.

Treatment Methods

The nanoparticle compositions as described herein are useful in treatingcancer cells and/or tumors in a mammal. In a preferred embodiment, themammal is a human (i.e., a human patient). Preferably, the lyophilizednanoparticle composition is reconstituted (suspended in an aqueousexcipient) prior to administration.

In one aspect is provided a method for treating a cancer cell, themethod comprising contacting the cell with an effective amount ofnanoparticle composition as described herein to treat the cancer cell.Treatment of a cancer cell includes, without limitation, reduction inproliferation, killing the cell, preventing metastasis of the cell, andthe like.

In one aspect is provided a method for treating a tumor in a patient inneed thereof, the method comprising administering to the patient atherapeutically effective amount of a nanoparticle composition asdescribed herein to treat the tumor. In one embodiment, the size of thetumor is reduced. In one embodiment, the tumor size does not increase(i.e. progress) for at least a period of time during and/or aftertreatment.

In one embodiment, the nanoparticle composition is administeredintravenously. In one embodiment, the nanoparticle composition isadministered directly to the tumor. In one embodiment, the nanoparticlecomposition is administered by direct injection or perfusion into thetumor.

In one embodiment, the method comprises:

-   -   a) administering the nanoparticle composition once a week for        three weeks;    -   b) ceasing administration of the nanoparticle composition for        one week; and    -   c) optionally repeating steps a) and b) as necessary to treat        the tumor.

In one embodiment, the therapeutically effective amount of thenanoparticles described herein comprises about 50 mg/m² to about 200mg/m² carrier protein or carrier protein and therapeutic agent. In apreferred embodiment, the therapeutically effective amount comprisesabout 75 mg/m² to about 175 mg/m² carrier protein or carrier protein andtherapeutic agent. Contemplated values include any value, subrange, orrange within any of the recited ranges, including endpoints.

In one embodiment, the therapeutically effective amount comprises about20 mg/m² to about 90 mg/m² antibody. In a preferred embodiment, thetherapeutically effective amount comprises 30 mg/m² to about 70 mg/m²antibody. Contemplated values include any value, subrange, or rangewithin any of the recited ranges, including endpoints.

Cancers or tumors that can be treated by the compositions and methodsdescribed herein include, but are not limited to: biliary tract cancer;brain cancer, including glioblastomas and medulloblastomas; breastcancer; cervical cancer; choriocarcinoma; colon cancer; endometrialcancer; esophageal cancer, gastric cancer; hematological neoplasms,including acute lymphocytic and myelogenous leukemia; multiple myeloma;AIDS associated leukemias and adult T-cell leukemia lymphoma;intraepithelial neoplasms, including Bowen's disease and Paget'sdisease; liver cancer (hepatocarcinoma); lung cancer; lymphomas,including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas;oral cancer, including squamous cell carcinoma; ovarian cancer,including those arising from epithelial cells, stromal cells, germ cellsand mesenchymal cells; pancreas cancer; prostate cancer; rectal cancer;sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma,fibrosarcoma and osteosarcoma; skin cancer, including melanoma, Kaposi'ssarcoma, basocellular cancer and squamous cell cancer; testicularcancer, including germinal tumors (seminoma, non-seminoma[teratomas,choriocarcinomas]), stromal tumors and germ cell tumors; thyroid cancer,including thyroid adenocarcinoma and medullar carcinoma; and renalcancer including adenocarcinoma and Wilms tumor. In importantembodiments, cancers or tumors include breast cancer, lymphoma, multiplemyeloma, and melanoma.

In general, the compounds of this invention will be administered in atherapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. The actualamount of the compound of this invention, i.e., the nanoparticles, willdepend upon numerous factors such as the severity of the disease to betreated, the age and relative health of the subject, the potency of thecompound used, the route and form of administration, and other factorswell known to the skilled artisan.

An effective amount of such agents can readily be determined by routineexperimentation, as can the most effective and convenient route ofadministration, and the most appropriate formulation. Variousformulations and drug delivery systems are available in the art. See,e.g., Gennaro, A. R., ed. (1995) Remington's Pharmaceutical Sciences,18th ed., Mack Publishing Co.

An effective amount or a therapeutically effective amount or dose of anagent, e.g., a compound of the invention, refers to that amount of theagent or compound that results in amelioration of symptoms or aprolongation of survival in a subject. Toxicity and therapeutic efficacyof such molecules can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., bydetermining the LD50 (the dose lethal to 50% of the population) and theED50 (the dose therapeutically effective in 50% of the population). Thedose ratio of toxic to therapeutic effects is the therapeutic index,which can be expressed as the ratio LD50/ED50. Agents that exhibit hightherapeutic indices are preferred.

The effective amount or therapeutically effective amount is the amountof the compound or pharmaceutical composition that will elicit thebiological or medical response of a tissue, system, animal or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician. Dosages may vary within this range depending upon the dosageform employed and/or the route of administration utilized. The exactformulation, route of administration, dosage, and dosage interval shouldbe chosen according to methods known in the art, in view of thespecifics of a subject's condition.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety that are sufficient to achieve thedesired effects; i.e., the minimal effective concentration (MEC). TheMEC will vary for each compound but can be estimated from, for example,in vitro data and animal experiments. Dosages necessary to achieve theMEC will depend on individual characteristics and route ofadministration. In cases of local administration or selective uptake,the effective local concentration of the drug may not be related toplasma concentration.

EXAMPLES

The present disclosure is illustrated using nanoparticles composed ofalbumin-bound paclitaxel (i.e., Abraxane®) or cisplatin as core, andbevacizumab (i.e., Avastin®) or Rituximab (i.e., Rituxan®) asantibodies.

One skilled in the art would understand that making and using thenanoparticles of the Examples are for the sole purpose of illustration,and that the present disclosure is not limited by this illustration.

Any abbreviation used herein, has normal scientific meaning. Alltemperatures are ° C. unless otherwise stated. Herein, the followingterms have the following meanings unless otherwise defined:

ABX = Abraxane ®/(albumin-bound paclitaxe AC = cisplatin-bound ABX ACN =acetonitrile ADC = antibody dependent chemotherapy BEV = bevacizumab BSA= bovine serum albumin dH2O = distilled water DMEM = Dulbecco's ModifiedEagle's Medium nM = nano molar EdU = 5-ethynyl-2′-deoxyuridine EM =electron microscopy FCB = flow cytometry buffer FITC = Fluorescein kD =kilo-dalton Kd = dissociation constant kg = kilogram KV = kilo-voltsL/hr = liter/hour LC-MS = liquid chromatography-mass spectrometry M =molar mCi = millicuries mg = milligram ml or mL = milliliter m² = squaremeters mm³ = cubic millimeter μg = microgram μl = microliter μm =micrometer/micron PBS = Phosphate buffered saline pK = pharmacokineticsRT = room temperate rpm = rotations per minute v = volts x g = timesgravity

Example 1 Nanoparticle Preparation

Abraxane (ABX) (10 mg) was suspended in bevacizumab (BEV) (4 mg [160 μl]unless otherwise indicated), and 840 μl of 0.9% saline was added to givea final concentration of 10 mg/ml and 2 mg/ml of ABX and BEV,respectively. The mixture was incubated for 30 minutes at roomtemperature (or at the temperature indicated) to allow particleformation. For Mastersizer experiments to measure particle size ofABX:BEV complexes, 10 mg of ABX was suspended in BEV at concentrationsof 0 to 25 mg/ml. Complexes of ABX with rituximab (0-10 mg/ml) ortrastuzumab (0-22 mg/ml) were formed under similar conditions.

For use in humans, the ABX:BEV complexes may be prepared by obtainingthe dose appropriate number of 4 mL vials of 25 mg/mL BEV and dilutingeach vial per the following directions to 4 mg/mL. The dose appropriatenumber of 100 mg vials of ABX can be prepared by reconstituting to afinal concentration containing 10 mg/mL ABX nanoparticles. Using asterile 3 mL syringe, 1.6 mL (40 mg) of bevacizumab (25 mg/mL) can bewithdrawn and slowly injected, over a minimum of 1 minute, onto theinside wall of each of the vials containing 100 mg of ABX. Thebevacizumab solution should not be injected directly onto thelyophilized cake as this will result in foaming. Then, using a sterile12 mL sterile syringe, 8.4 mL 0.9% Sodium Chloride Injection, USP, canbe withdrawn and slowly injected, over a minimum of 1 minute, 8.4 mLonto the inside wall of each vial containing ABX 100 mg and BEV 40 mg.Once the addition of BEV 1.6 mL and 0.9% Sodium Chloride Injection, USP8.4 mL is completed, each vial can be gently swirled and/or invertedslowly for at least 2 minutes until complete dissolution of anycake/powder occurs. Generation of foam should be avoided. At this point,the concentration of each vial should be 100 mg/10 mL ABX and 40 mg/10mL BEV. The vials containing the ABX and BEV should sit for 60 minutes.The vial(s) should be gently swirled and/or inverted every 10 minutes tocontinue to mix the complex. After 60 minutes has elapsed, thecalculated dosing volume of ABX and BEV should be withdrawn from eachvial and slowly added to an empty viaflex bag. An equal volume of 0.9%Sodium Chloride Injection, USP is then added to make the finalconcentration of ABX 5 mg/mL and BEV 2 mg/mL. The bag should then begently swirled and/or inverted slowly for 1 minute to mix. The ABX:BEVnanoparticles can be stored for up to 4 hours at room temperaturefollowing final diluation.

Example 2 Binding of ABX and BEV In Vitro

To determine whether ABX and BEV interact, the nanoparticles formed inExample 1 were analyzed by flow cytometry and electron microscopy.

Methods

Flow Cytometry:

AB160 was produced as described in Example 1 above. To determine bindingof BEV to ABX, visualization of AB 160 was performed on an Accuri C6flow cytometer (BD Franklin Lakes, N.J.) and data analysis was doneusing Accuri C6 software. Biotinylated (5 μg) goat anti-mouse IgG(Abeam, Cambridge, Mass.) was labeled with 5 μg of streptavidin PE(Abeam, Cambridge, Mass.). The goat anti-mouse IgG was chosen to labelAB160 because the Fab portion of the BEV is mouse derived. ABX and AB160were incubated with the PE-labeled goat anti-mouse IgG for 30 minutes atroom temperature, washed and visualized by flow cytometery.

Electron Microscopy:

Five μl ABX, dissolved in PBS at 6 mg/ml, was added to a 300-meshparlodian-carbon coated copper grid and allowed to sit for 1 minute. Apointed piece of filter paper was touched to the drop to remove excessliquid, leaving a thin film on the grid. The grids were allowed to dry.To dissolve the buffer crystals left on the dried grid, the sample waswashed three times in dH2O. A small drop of 1% phosphotungstic acid(PTA), pH 7.2, was added to the grid. The grid was then again touched bya pointed piece of filter paper to remove excess liquid, leaving a thinfilm on the grid and allowed to dry. BEV (Genentech) at 25 mg/ml in 0.9%sodium chloride solution was diluted with PBS at 1:10 ratio. Five μl ofBEV was loaded on nickel formvar-coated grid and allowed to air dry for30 minutes to 1 hour. For the AB160, 10 mg/ml ABX, dissolved in PBS, and4 mg/ml BEV, in 0.9% sodium chloride solution, were mixed at 2.5:1ratio. The complex was further diluted with PBS at 1:5. Five μl of thecomplex was loaded on nickel formvar-coated grid and air dried for 30minutes to 1 hour. Both samples were incubated for 1 hour in goatanti-mouse IgG with 6 nm gold-conjugated particles (Electron MicroscopySciences), diluted 1:30 with 10% FCB/PBS, washed 6 times with PBS (each2 minutes), 6 times with dH₂O, then stained with the mixture of 2%methylcellulose and 4% UA (9:1) for 5 minutes. Filter paper was used todrain the stain and the grid was air dried for 1 hour. Both samples wereincubated overnight in donkey anti-mouse IgG with 6 nm gold-conjugatedparticles (Jackson ImmunoResearch) diluted 1:25 with 10% FCB/PBS, washed6 times with PBS (each 2 minutes), 6 times with dH2O water, stained with1% PTA for 5 minutes, air dried, covered with 2% methylcellulose, andair dried for 1 hour. The micrographs were taken on a JEOL1400 atoperating at 80 KV.

Results

ABX (10 mg/ml) was co-incubated with 4 mg/ml BEV in vitro and found thatthey formed 160 nm nanoparticles (referred to herein as AB160). Becausethe Fab portion of the IgGI (BEV) is of mouse origin, particlescontaining BEV were selectively labeled with purified goat anti-mouseIgG followed by anti-goat PE as a secondary antibody. As a negativecontrol, samples were stained with the anti-goat PE only. Particles werevisualized by flow cytometry and demonstrated a bright signal ofanti-mouse IgGI binding to AB 160 (41.2% positive) relative to ABX (6.7%positive) alone (FIG. 1A). To validate binding of BEV to ABX, the BEVwere labeled with gold-labeled mouse anti-human IgG and the particleswere visualized with electron microscopy (FIG. 1B). Surprisingly, the EMpictures suggest a monolayer of BEV surrounding ABX nanoparticles.

To determine what protein (albumin or BEV) the paclitaxel remains boundto when the complex breaks down, AB160 were made and collectedfractions: the particulate (nanoAB160), proteins greater than 100 kD andproteins less than 100 kD. Paclitaxel was measured in each fraction byliquid chromatography-mass spectrometry (LC-MS). Roughly 75% of thepaclitaxel remained within the particulate, and the majority of theremaining paclitaxel was associated with the fraction containingproteins 100 kD or greater (FIG. 1 C, top), suggesting that when theparticulate dissociates the paclitaxel is bound to BEV alone or a BEVand albumin heterodimer. This indicates that the dissociated complexescontain the chemotherapy drug with the antibody, which would stilltraffic to the high-VEGF tumor microenvironment. These findings wereconfirmed by Western blot analysis of the supematants from AB 160, whichshowed that BEV and paclitaxel co-localize at approximately 200 kD, asize consistent with a paclitaxel-BEV-albumin protein complex (FIG. 1 C,bottom).

Example 3 Function of AB160 In Vitro

Confirmation that the two key elements in the complexes, the antibod andthe paclitaxel, retained their function when present in the complexeswas demonstrated.

Methods

In Vitro Toxicity:

The A375 human melanoma cell line (ATCC Manassa, Va.) and Daudi B-celllymphoma line (ATCC Manassa, Va.) were cultured in DMEM with 1% PSG and10% FBS. Cells were harvested and plated at 0.75×10⁶ cells per well in24 well plates. Cells were exposed to ABX or AB160 at paclitaxelconcentrations from 0 to 200 μg/ml overnight at 37° C. and 5% CO₂. Tomeasure proliferation, the Click-iT EdU (Molecular Probes, Eugene,Oreg.) kit was utilized. Briefly, 10 mM EdU was added to the wells andincubated overnight with the cells and ABX or AB160. The cells werepermeabilized with 1% saponin and intercalated EdU was labeled with aFITC-conjugated antibody. The proliferation index was determined bydividing the FITC positive cells from each treatment by the maximumproliferation of untreated EdU labeled cells.

VEGF ELISA:

To determine whether BEV can still bind its ligand, VEGF, when bound toABX, a standard VEGF ELISA (R and D Systems, Minneapolis, Minn.) wasemployed. AB 160 was prepared as described and 2000 pg/ml VEGF was addedto the AB 160 complex or ABX alone. The VEGF was incubated with thenanoparticles for 2 hours at room temperature. The suspension was spunat 6000 rpm for 15 minutes, supernatants were collected and free VEGFwas measured by ELISA. Briefly, ELISA plates were coated with captureantibody overnight at 4° C. Plates were washed, blocked and standardsand samples were added. After washing, detection antibody was added andplates were developed with substrate (R and D Systems, Minneapolis,Minn.). Absorbance was measured at 450 nm using a Versamax ELISA platereader (Molecular Devices, Sunnyvale, Calif.). The concentration ofunbound VEGF was determined with a standard curve from 0 to 2000 pg/ml.

Results

AB 160 has similar toxicity relative to ABX alone in an in vitrotoxicity assay with the human melanoma cell line, A375, suggesting thatthe paclitaxel functions equally in either formulation (FIG. 1D).

To test the binding of VEGF to BEV in the AB160 complex, AB160 or ABXwas co-incubated with VEGF, the particulate removed, and the supernatanttested for VEGF content. The lack of VEGF in the supernatant measuredfrom AB160 (<10% VEGF unbound) indicated that the VEGF was bound by theBEV in the AB 160 complex, while it remained free when incubated withthe ABX (>80% VEGF unbound) alone (FIG. 1 E).

Importantly, these assays demonstrated that the paclitaxel in AB160retains its toxicity to tumor cells and the bound BEV maintains theability to bind its ligand, VEGF.

Example 4 Particle Size and Protein Affinity

To understand the characteristics of the nanoparticles formed whenbinding BEV to ABX, the size of the ABX:BEV complexes was determinedrelative to ABX.

Methods

Mastersizer and Nanosight:

The particle size of ABX and antibody-ABX drug complexes were measuredby dynamic light scattering on a Mastersizer 2000 (Malvern Instruments,Westborough, Mass.). To measure particle size, 2 ml (5 mg/ml) ofAbraxane or complex was added to the sample chamber. Data were analyzedwith Malvern software and particle size distributions were displayed byvolume. The particle sizes and stability were later validated using theNanosight System (Malvern Instruments, Westborough, Mass.). The ABX orcomplex particles were diluted to the appropriate range to accuratelymeasure particle sizes. Data was displayed by particle sizedistribution; however, the nanoparticle tracking analysis uses Brownianmotion to determine particle size.

Binding Assay:

Biotinylated BEV, rituximab or trastuzumab at 100 μg/ml was bound to thestreptavidin probe (ForteBio Corp. MenloPark, Calif.). The binding ofABX was measured by light absorbance on the BLitz system (ForteBio Corp.MenloPark, Calif.) at 1000, 500 and 100 mg/ml. The association anddissociation constants were calculated using the BLItz software.

Bio-Layer Interferometry (BLItz) technology was utilized to assess thebinding affinity of BEV to ABX. Biotinylated BEV was bound to thestreptavidin probe and exposed to ABX (1000, 500, and 100 μg/ml). Thedissociation constant (Kd) of BEV and ABX is 2.2×10⁻⁸ M at roomtemperature and pH 7, consistent with a strong non-covalent interaction.The binding affinity of BEV and ABX is within the range of dissociationconstants observed between albumin and natural or engineeredalbumin-binding domains of some bacterial proteins. Nilvebrant, J. etal. (2013) Comput Struct Biotechnol J 6:e201303009.

Results

ABX:BEV nanoparticles were consistently larger (approximately 160 nm)than the 130 nm ABX alone (FIG. 2a ). The size of the nanoparticlecreated directly correlated to the concentration of BEV used, withmedian sizes ranging from 0.157 to 2.166 μm. (FIG. 2A). With the goal ofthese studies being a Phase I clinical trial, the smallest ABX:BEVparticle (AB160) were focused on because it is the most similar to the130 nm ABX. The size of the AB 160 particle was consistent with ABX plusa monolayer of BEV surrounding it and with the EM image of the particle(see FIG. 1B).

To determine whether intravenous administration conditions affectnanoparticle size distributions, the particle size distributions ofAB160 (or ABX) incubated in saline for up to 24 hours at roomtemperature were evaluated. AB160 size distribution does notsignificantly change for up to 24 hours (FIGS. 9A and 9B). However, by 4hours at room temperature, there is some evidence of AB160 breakdown byELISA (FIG. 9C).

To determine the stability of AB160 in plasma, ABX or AB160 wasincubated in saline or heparinized human plasma at relative volumeratios of 9:1 or 1:1. Notably, no particles (0.01 to 1 μm) were detectedwhen either ABX (FIG. 10, top panel) or AB160 (FIG. 10, bottom panel) isincubated in plasma at equal volumes (1:1).

Western blot (data not shown) indicated that, in saline or heparinizedhuman plasma, the AB160 dissociated into smaller protein conjugates thatstill contain the tumor-targeting antibody, albumin and the cytotoxicagent, paclitaxel. These protein conjugates retain their ability totarget the tumor and, once at the tumor site, can quickly dissolve andrelease the cytotoxic payload to effectively initiate tumor regressionwithout internalization of the entire nanoparticle by tumor cells.

Next, the ABX was suspended in BEV and the mixture diluted with salineat pH 3, 5, 7, or 9 prior to incubation at various temperatures (RT, 37°C. and 58° C.) to allow particle formation in order to test whetherbinding affinity was pH- and/or temperature-dependent. The bindingaffinity of ABX and BEV is both pH- and temperature-dependent, with thehighest binding affinity observed when the particles are formed at pH 5and 58° C. (FIG. 2B).

To determine if the higher affinity binding of BEV and ABX at 58° C. andpH 5 translated into stability of the complex, various preparations werecompared by nanoparticle tracking analysis (Nanosight). The stability ofAB160 prepared at 58° C. and pH 5 (AB1600558), room temperature and pH 7(AB16007), or 58° C. and pH 7 (AB1600758) was compared to ABX exposed tothe same conditions (ABX0558, ABX07, and ABX0758, respectively) afterincubation in human AB serum for 0, 15, 30, or 60 minutes.

The particles made under higher affinity conditions (pH 7 and 58° C.)were also more stable, as indicated by the number of particles presentper mg ABX after exposure to human AB serum. The AB160 particlesexhibited increased stability in human serum that correlated with theirbinding affinities. In particular, AB16007 and AB1600558 were morestable in both saline and human serum than ABX alone, as determined bysize and number of particles measured per mg ABX (FIG. 2C and Table 3).This shows that the stability of AB160 particles can be manipulated bychanging the conditions under which the AB160 particles are formed.

TABLE 3 Stability of AB160 and ABX in human AB serum Human AB SerumSaline 0 min 15 min 30 min 60 min ABX07 221.5 54.4 85.2 84 32.1 AB160072500 516 508 756 296 ABX0758 236 182.4 155.4 54 66 AB1600758 2460 436236 260 176 ABX0558 348 510 86.8 90 64 AB1600558 7296 2200 1224 1080 960Particles per mg ABX × 10⁻⁸

These data demonstrated that BEV binds to ABX with affinity in thepicomolar range, indicating a strong non-covalent bond, and demonstrateda particle size distribution consistent with ABX surrounded by amonolayer of antibody molecules; the size of the particles created isdependent on the antibody concentration.

Example 5 Efficacy of AB160 in Mice

A xenograft model of A375 human melanoma cells implanted into athymicnude mice was employed to test AB 160 efficacy in vivo.

Methods

In vivo experiments were performed at least 2 times. The number of micerequired for those experiments was determined by power analysis. Mousetumors were measured 2-3 times/week and mice were sacrificed when thetumor was 10% by weight. Mice that had complete tumor responses weremonitored for 60-80 days post-treatment. The end point of the mousestudies was median survival. Kaplan-Meier curves were generated andMantle-Cox test was performed to determine significance of mediansurvival between treatment groups. The in vitro results presented arerepresentative of at least 5 repeated experiments. Statistical analysesof in vitro and in vivo percent change from baseline experiments weredone using the Student's t-test.

Mouse Model:

To test tumor efficacy, 1×10⁶ A375 human melanoma cells were implantedinto the right flank of athymic nude mice (Harlan Sprague Dawley,Indianapolis, Ind.). When the tumors had reached a size of about 700mm³, the mice were randomized and treated with PBS, ABX (30 mg/kg), BEV(12 mg/kg), BEV followed by ABX, or AB160 at the above concentrations.For the mouse experiments testing bigger AB particles, the highest doseof BEV (45 mg/kg) necessary to create the larger particles was used inthe BEV-only treatment group. Tumor size was monitored 3 times/week andtumor volume was calculated with the following equation:(length*width²)/2. Mice were sacrificed when the tumor size equaled 10%of the mouse body weight or about 2500 mm³. The day 7 percent changefrom baseline was calculated as follows: [(tumor size on treatmentday-tumor size on day 7)/tumor size on treatment day]*100. The in vivotesting of the AR160 was similar except 5×10⁶ Daudi cells were injectedinto the right flank of athymic nude mice.

Results

AB160 was tested relative to PBS, the single drugs alone, and the drugsadministered sequentially. Mice treated with AB160 had significantlyreduced tumor size compared to all other treatment groups (p=0.0001 to0.0089) at day 7 post-treatment, relative to baseline (FIG. 3A). Tumorsin all of the mice treated with AB160 had regressed at day 7, and thistumor response translated into significantly increased median survivalof the AB 160 group relative to all other groups (FIG. 3B), with asurvival of 7, 14, 14, 18 and 33 days for the PBS (p<0.0001), BEV(p=0.003), ABX (p=0.0003), BEV+ABX (p=0.0006) and AB160 groups,respectively.

It is likely that larger tumors have a higher local VEGF concentration.When data were analyzed based on the size of the tumor on day oftreatment (<700 mm³ and >700 mm³), the larger tumors were shown to havea greater response to AB 160, suggesting that higher tumor VEGFconcentration attracts more BEV-targeted ABX to the tumor. Thedifference in the percent change from baseline was significant for theAB160 groups (p=0.0057). This observation was not seen in the ABX only(p=0.752) group, where the ABX has no targeting capability (FIG. 3C).

Particles of increasing size were prepared using increasing BEV:ABXratios as shown in FIG. 2A. Tumor regression and median survivalpositively correlated with increasing particle size, indicating thatbiodistribution of larger particles may be altered relative to thesmaller ones (FIGS. 3D and 3E). Full toxicity studies were performed onthe mice and no toxicities were noted.

Example 6 Paclitaxel Pharmakokinetics in Mice

To compare the pharmacokinetics (pk) of AB160 and ABX, plasma paclitaxelconcentrations were measured in mice administered AB160 or ABX at 0, 4,8, 12 and 24 hours.

Methods

Paclitaxel Pharmacokinetics:

The liquid chromatographic separation of paclitaxel and d5 paclitaxelwere accomplished using an Agilent Poroshell 120 EC-C18 precolumn (2.1×5mm, 2.7 μm, Chrom Tech, Apple Valley, Minn.) attached to an AgilentPoroshell 120 EC-C18 analytical column (2.1×100 mm, 2.7 μm Chrom Tech,Apple Valley, Minn.) at 40° C., eluted with a gradient mobile phasecomposed of water with 0.1% formic acid (A) and ACN with 0.1% formicacid (B) with a constant flow rate of 0.5 ml/minute. The elution wasinitiated at 60% A and 40% B for 0.5 minutes, then B was linearlyincreased from 40-85% for 4.5 minutes, held at 85% B for 0.2 minutes,and returned to initial conditions for 1.3 minutes. Autosamplertemperature was 10° C. and sample injection volume was 2 μl. Detectionof paclitaxel and the internal standard d5-paclitaxel were accomplishedusing the mass spectrometer in positive ESI mode with capillary voltage1.75 kV, source temp 150° C., desolvation temp 500° C., cone gas flow150 L/hr, desolvation gas flow 1000 L/hr, using multiple reactionmonitoring (MRM) scan mode with a dwell time of 0.075 seconds. The conevoltages and collision energies were determined byMassLynx-Intellistart, v4.1, software and varied between 6-16 V and12-60 eV, respectively. The MRM precursor and product ions weremonitored at m/z 854.3>105.2 for paclitaxel and 859.3>291.2 for d5paclitaxel. The primary stock solutions of paclitaxel (1 mg/ml in EtOH)and d5 paclitaxel (1 mg/ml in EtOH) were prepared in 4 ml ambersilanized glass vials and stored at −20° C. Working standards wereprepared by dilution of the stock solution with ACN in 2 ml ambersilanized glass vials and stored at −20° C. Plasma samples wereextracted as follows, 100 μl plasma sample was added to a 1.7 mlmicrocentrifuge tube containing d5 paclitaxel (116.4 nM or 100 ng/ml)and 300 μl ACN, vortexed, incubated at room temperature for 10 minutesto precipitate proteins, and centrifuged (14,000 rpm) or 3 minutes. Thesupernatant was filtered on an Agilent Captiva ND^(lipids) plate (ChromTech, Apple Valley, Minn.), collected in a deep 96-well plate, and driedusing nitrogen gas. The samples were reconstituted using 100 μl ACN andshaken on a plate shaker (high speed) for 5 minutes. Plasma standardcurves were prepared daily containing paclitaxel (0.59-5855 nM or0.5-5000 ng/ml) and d5 paclitaxel (116.4 nM) for paclitaxelquantitation. Mouse tumors were thawed on ice, weighed, and diluted 2parts (weight to volume) in 1×PBS. Tumors were then homogenized using aPRO200 tissue homogenizer using the saw tooth probe (5 mm×75 mm). Tumorhomogenate was than processed the same as the human plasma samples.

Mouse Imaging:

Avastin and IgG control solutions were prepared and 1-125 labeled perprotocol (Imanis Life Sciences). Briefly, Tris Buffer (0.125 M Tris-HCl,pH 6.8, 0.15 M NaCl) and 5 mCi Na¹²⁵ I were added directly to iodinationtubes (ThermoFischer Scientific, Waltham, Mass.). The iodide was allowedto activate and was swirled at room temperature. Activated iodide wasmixed with the protein solution. 50 μl of Scavenging Buffer (10 mgtyrosine/mL in PBS, pH 7.4) was added and incubated for five minutes.After addition of Tris/BSA buffer and mixing, samples were applied in 1OK MWCO dialysis cassettes against pre-cooled PBS for 30 minutes, 1hour, 2 hours, and overnight at 4° C. Radioactivity was determined byGamma counter, then disintegrations per minute (DPM) and specificactivity were calculated. Mice were injected in their tail vein withAvastin I-125, Abraxane-Avastin I-125, Abraxane-human IgG I-125, orAbraxane only. Animals were imaged at 3, 10, 24 and 72 hourspost-administration via SPECT-CT imaging using the U-SPECT-II/CT scanner(MILabs, Utrecht, The Netherlands). SPECT reconstruction was performedusing a POSEM (pixelated ordered subsets by expectation maximization)algorithm. CT data were reconstructed during the Feldkamp algorithm.Co-registered images were further rendered and visualized using PMODsoftware (PMOD Technologies, Zurich, Switzerland). Animals weresacrificed and dissected at 72 hours post-injection. Selected tissuesand organs of interest were measured using radioisotope dose calibrator(Capintec CRC-127R, Capintec Inc.).

Results

Results of the first pk experiment are provided in FIGS. 4A and 4B. Thearea under the curve (AUC) and maximum serum concentration (C_(max))were calculated in A375 tumor bearing and non-tumor bearing mice. In thefirst pk experiment the C_(max) and AUC were very similar in thenon-tumor bearing mice for AB160 and ABX (63.3+/−39.4 vs. 65.5+/−14.4and 129 vs. 133 μg/ml, respectively). However, in the tumor bearingmice, the C_(max) and AUC for the treatment groups were different(55.7+/−21.2 vs 63.3+/−17.3 and 112 vs 128 μg/ml, respectively) (FIG.4C). Although this difference was not statistically significant, it isconsistent with superior targeting by AB160, relative to ABX.

A second pk experiment was performed with additional early time pointsand large versus small tumor sizes (FIGS. 4D-4F). The results of thisexperiment demonstrated smaller AUC in tumor bearing mice relative tonon-tumor bearing mice, with the lowest blood values of paclitaxel inthe large tumor mice relative to the small tumor mice (80.4+/−2.7,48.4+/−12.3, and 30.7+/−5.2 for ABX-treated non-tumor, small tumor andlarge tumor bearing mice, respectively; 66.1+/−19.8, 44.4+/−12.1 and22.8+/−6.9 for AB160-treated). Similarly, the C_(max) dropped in bothtreatment groups in mice with larger tumors (47.2, 28.9 and 19.7 μg/mlfor ABX and 40.1, 26.9 and 15.3 μg/ml for AB160) (FIG. 4G). The AUC andC_(max) of paclitaxel in blood were lower in AB 160-treated micerelative to ABX-treated mice. Although not statistically significant,this data is further consistent with higher deposition of paclitaxel inthe tumors treated with AB160.

To directly test this hypothesis, tumor paclitaxel concentrations byLC-MS were measured. The tumor paclitaxel concentration wassignificantly higher in tumors treated with AB160 relative to ABX at the4 hour (3473 μg/mg of tissue+/−340 vs 2127 μg/mg of tissue+/−3.5;p=0.02) and 8 hour (3005 μg/mg of tissue+/−146 vs 1688 μg/mg oftissue+/−146; p=0.01) time points, suggesting paclitaxel stays in thetumor longer when targeted by the antibody (FIG. 4H). This explains theblood pk and is consistent with redistribution of drug to tissuesincluding the tumor.

Live in vivo imaging of 1-125 labeled AB160 (Abx-AvtI125) and IgGisotype bound ABX (Abx-IgGI125) confirmed the results of the LC-MS, withhigher levels of 1-125 in the tumor of mice treated with AB160 relativeto IgG-ABX at 3 hours (32.2 uCi/g+/−9.1 vs 18.5 uCi/g+/−1.65; p=0.06)and 10 hours (41.5 uCi/g+/−6.4 vs 28.7 uCi/g+/−2.66; p=0.03) postinjection (FIGS. 41 and 4J). Taken together, these data demonstrate thatbinding BEV to ABX alters blood pk, and this alteration is due to aredistribution of the drug to the tumor tissue as shown by both LC-MS ofpaclitaxel and 1-125 labeling of BEV relative to an isotype matchedIgG1.

Without being bound by theory, it is believed that by binding atumor-targeted antibody to ABX, the pk is altered more dramatically thanABX alone, lowering the C_(max) and AUC in the blood because ofredistribution of AB160 to the tumor tissue. These results from mouseblood paclitaxel pk, tumor tissue levels of paclitaxel, and 1-125radioactivity levels in mice treated with AB160 relative to ABX alonesuggest that antibody targeting of the ABX alters biodistribution ofpaclitaxel such that increased levels reach the tumor and are retainedthere for a longer period of time, yielding enhanced tumor regression.

Example 7 Binding of Other Therapeutic Antibodies

The binding of the anti-human CD20 antibody (rituxamab) and theanti-HER2/neu receptor antibody (trastuzumab) to ABX was tested todetermine if other IgG therapeutic antibodies also exhibit binding toABX when combined ex vivo.

Methods

Nanoparticles containing rituximab or trastuzumab were prepared andtested as described in the above examples.

Results

The particle size of the complexes with both BEV and trastuzumab (HER)were very similar, with average sizes ranging from 0.157 to 2.166 μm(FIG. 2A) and 0.148 to 2.868 μm (FIG. 5B), respectively. In contrast,particles formed with rituximab became much larger at lower antibody:ABXratios, with particle sizes ranging from 0.159 to 8.286 μm (FIG. 5A).

The binding affinities of rituximab and trastuzumab with ABX weredetermined by BLitz under variable pH. Both antibodies bind withrelatively high affinity in the picomolar range (FIG. 5C). The rituximabaffinity to ABX decreased with higher pH, but trastuzumab affinity toABX was unaffected by pH (FIG. 5C).

The efficacy of the 160 nm particle made with rituximab (AR160) wastested in vitro and in vivo. In vitro, the B-cell lymphoma cell lineDaudi was treated with AR160, ABX, or rituximab alone at increasingconcentrations (0 to 200 μg/ml) of paclitaxel. AR160 (IC₅₀=10 μg/ml)significantly inhibited proliferation of Daudi cells treated for 24hours (p=0.024) compared to either ABX (IC₅₀>200 μg/ml) or rituximab(IC₅₀>200 μg/ml) alone (FIG. 6A).

In vivo, a xenotransplant model of Daudi cells was established inathymic nude mice. Once tumors were established, mice were treated withPBS, ABX, rituximab, ABX and rituximab given sequentially, or AR160. Onday 7 post treatment, tumors were measured and the percent change intumor size from baseline was calculated. AR160-treated tumors regressedor remained stable, while tumors in all other treatment groupsprogressed (FIG. 6B). The percent change from baseline tumor size in theAR160 group compared to all other groups was significant (p<0.0001). Themice treated with AR160 had a significantly longer median survival ofgreater than 60 days compared to 12, 16, and 12 days for mice treatedwith PBS (p<0.0001), ABX (p<0.0001), or rituximab (p=0.0002),respectively (FIG. 6C). However, the difference in median survival wasnot significant between AR160 and the sequentially treated groups(p=0.36). This may be because the rituximab binds to the tumor cells andremains on the cell surface, allowing the subsequently-administered ABXto bind to the antibody when it enters the tumor site, unlike BEV whichbinds a soluble target and not a cell surface marker.

Example 8 Binding of Other Chemotherapy Drugs to AB160

The efficacy of other chemotherapy drugs to form functionalnanoparticles was evaluated.

Methods

Nanoparticles containing cisplatin were prepared and tested as describedin the above examples.

Results

To test if another chemotherapy drug could bind to the AB160 particles,cisplatin and ABX were co-incubated and the amount of free cisplatinremaining in the supernatant was measured by HPLC. Approximately 60%(i.e., only 40% remains in the supernatant) of the cisplatin bound tothe ABX (FIG. 7A).

Next, tumor toxicity of AC relative to ABX and cisplatin alone wastested using A375 cells. The complexes were centrifuged to remove highlytoxic unbound cisplatin, and reconstituted in media to ensure that anyadditional toxicity of AC relative to ABX is due only to ABX-boundcisplatin. For parity, the ABX only was centrifuged in a similar manner.AC (IC₅₀=90 μg/ml) inhibited proliferation of A375 cells to a greaterextent than ABX alone (IC₅₀>1000 μg/ml) (FIG. 7B). The diminishedtoxicity in this experiment relative to other toxicity experiments isdue to some loss of drug in the centrifugation step, but the comparisonof ABX to AC remains relevant.

To determine the tumor toxicity of cisplatin-containing AB160 complexes,AB160 was co-incubated with cisplatin to form cisplatin containingparticles (ABC complex). The ABC complex was tested in the A375 melanomaxenotrasplant model relative to each drug alone and AB160. Tumorstreated with AB160, AB160+cisplatin given sequentially, and the ABCcomplex all showed regression in tumor size at 7 days post treatment(FIG. 7C), but the ABC complex conferred the longest median survival (35days, relative to AB160 and AB160+cisplatin at 24 and 26 days,respectively). Although the difference was not statistically significant(p=0.82 and 0.79) (FIG. 7D), the data is consistent with benefits of theABC complex to long-term survival rates.

These data demonstrated that the albumin portion of the ABX provides aplatform for other therapeutic antibodies to bind, such as rituximab andtrastuzumab, as well as other chemotherapy agents (e.g., cisplatin),which all had similar efficacy in vitro and in vivo as AB160.

Together these data demonstrate a simple way to construct a versatilenano-immune conjugate, which allows multiple proteins or cytotoxicagents to be bound to a single albumin scaffold. Improved efficacy ofthe targeted drug relative to the single agents alone was demonstratedin the mouse model, which is at least in part due to altered pk of theantibody-targeted drug. Furthermore, without being bound by theory, itis believed that the versatility of the presently disclosurednano-immune conjugate that does not require a linker or target cellinternalization will overcome the obstacles faced by other nanomedicinesin translating results from mice to humans.

Example 9 Lyophilization of AB160

AB160 was synthesized by adding 8 mg (3200) of bevacizumab to 20 mg ofAbraxane. 1.66 ml of 0.9% saline was then added for a final volume of 2ml for a final concentration of 4 mg/ml bevacizumab and 10 m g/mlAbraxane, and the mixture was allowed to incubate at room temperaturefor 30 minutes in a 15 ml polypropylene conical tube.

After the 30 minute room temperature incubation, the mixture was diluted1:2 in 0.9% saline to 2 mg/ml and 5 mg/ml bevacizumab and Abraxane,respectively. These are the concentrations of the 2 drugs when preparedby the pharmacy for administration to patients.

AB 160 was divided into twenty 200 μl aliquots in 1.5 ml polypropyleneeppendorfs and frozen at −80° C.

Once frozen, the aliquots were lyophilized overnight with the Virtis 3Lbenchtop lyophilizer (SP Scientific, Warmister, Pa.) with therefrigeration on. A lyophilized preparation was generated.

The dried aliquots were stored at room temperature in the same 1.5 mlpolypropylene eppendorfs. These samples were readily reconstituted insaline at room temperature for 30 minutes, followed by centrifugationfor 7 minutes at 2000×g. The resulting sample was then resuspended inthe appropriate buffer, as needed.

By comparison, a sample that was dried with heat and a speed vacuum wasimpossible to reconstitute.

Example 10 Testing of Lyophilized Preparations

Samples were reconstituted at different time points after lyophilizationand tested for their physical properties against ABX, and freshly madeAB160.

Particle size distribution was evaluated as described above.

VEGF binding was evaluated by incubation of the sample with VEGF for 2hours at room temperature, centrifuged at 2000×g for 7 minutes. Theamount of VEGF bound to the pellet (corresponding to the nanoparticles)or remaining in the supernatant was measured with ELISA.

Paclitaxel activity was assessed by cytotoxicity against A375 cells invitro.

Surprisingly, lyophilization did not significantly affect either theparticle size, VEGF binding, or the activity of paclitaxel as shown bythe ability to inhibit cancer cell proliferation. This result held forlyophilized samples stored for 1 month (FIGS. 8A-8C) or 10 months (FIGS.8D-8F).

Further surprising is that these results were observed withnanoparticles lyophilized without the use of cryoprotectants or otheragents that may adversely effect human therapeutic use.

Example 11 Efficacy of AB160 in Humans

AB160 was tested in a phase 1, first-in-man, clinical trial testing thesafety of AB160 administered to patients with metastatic malignantmelanoma that have failed prior therapies. The study utilizes aclassical 3+3, phase 1 clinical trial design, testing 3 different dosesof AB160 in the following schema:

TABLE 4 Dose AB-complex Both drugs MUST be reduced Level ABX doseAccompanying BEV dose 3 175 mg/m² 70 mg/m² 2 150 mg/m² 60 mg/m²  1* 125mg/m² 50 mg/m² −1  100 mg/m² 40 mg/m² −2   75 mg/m² 30 mg/m² *Dose level1 refers to the starting dose.

The doses were selected relevant to doses of Abraxane currently used inclinical practice. AB160 was made prior to each treatment dose.Treatments were administered as a 30 minute intravenous infusion on days1, 8 and 15 of a 28-day treatment cycle. Treatments were continued untilintolerable toxicity, tumor progression or patient refusal. Prior toevery treatment cycle, patients were evaluated for toxicity; tumorevaluations were performed every other cycle (RECIST).

The study is accompanied by formal (in-patient) pharmacokinetic studiesassociated with dose 1 of cycles 1 and 2 of therapy.

Five patients have been administered AB160, at 100 mg/m² of ABX and 40mg/m² of BEV, of which four have been analyzed.

TABLE 5 Disease course in Phase I study Disease Course: Dose Level 100mg/m² off, number treatment follow-up Patient of cycles response PFStime reasons time 1 8 stable 238  off, 444+ progression 2 6 stable 400+off, toxicity 400+ 3 1 — 182+ off, toxicity 182+ 4 6 stable 181  off,203+ progression

PFS refers to median progression free survival, i.e. the number of daysof treatment before the cancer recurred. Adverse events are listedbelow. There was no dose limiting toxicity (DLT), i.e. the adverseevents were not linked to the dose of AB 160. More detail is provided inTable 6.

TABLE 6 Adverse events in Phase I study patient toxicity DLT 1 grade 2lymphopenia NO 2 grade 3 neutropenia and leukopenia NO grade 2 3 grade 2colonic perforation, fatigue, and blood NO bilirubin increase 4 grade 2neutropenia NO

TABLE 7 Treatment Course: Dose Level 100 m/m² number of cycles number ofcycles number of cycles where day dose where dose reason for cycleswhere day 15 reasons day reductions reduction dose patient administered15 omitted omitted 15 omitted taken taken reductions status 1 8 0 1 4grd 2 off, sensory progression neuropathy 2 6 3 1, 2, 4 grd 3 2 3, 5cycle 3: grade off toxicity neutropenia and 3 neutropenia persistent grdleukopenia-all 3 and leukopenia 2 sensory cycles cycle 5: grade 3neuropathy neutropenia, leukopenia, and fatigue and grd 2 sensoryneuropathy 3 1 off toxicity grd 2 colonic perforation 4 6 2 3, 5 grd 2sensory off, neuropathy-both progression cycles

The mean PFS was 7.6 months and the median was 7.0 months.

Comparison with Other Clinical Trials

The following table shows other published clinical studies for taxanetherapy for metastatic melanoma.

TABLE 8 Taxane therapy for metastatic melanoma Study or Author N Rxregimens PFS OS Hauschild 135 C = AUC 6 (q21) 4.5 10.5 P = 225 mg/m2; Dl(q21) Flaherty 411 C = AUC 6 (q21) 4.9 11.3 P = 225 mg/m2; Dl (q21)N057E 41 C = AUC2; Dl, 8 15 (q28) 4.5 11.1 35 A = 100 mg/m2; Dl, 8, 15(q28) 4.1 10.9 N047A 53 C = AUC 6; Dl (q28) 6.0 12.0 P = 80 mg/m2; Dl,8, 15 (q28) B = 10 mg/kg; Dl, 15 (q28) BEAM 71 C = AUC5; Dl (q21) 4.28.6 P = 175 mg/m2; Dl (q21) 143 C = AUC5; Dl (q21) 5.6 12.3 P = 175mg/m2; Dl (q21) B = 15 mg/kg; Dl (q21) N0775 51 C = AUC6 (5); Dl (q28)6.2 13.9 A = 100 (80) mg/m2; Dl, 8, 15 (q28) B = 10 mg/kg; Dl, 15 (q28)Spitler 50 A = 150 mg/m2; Dl, 8, 15 (q28) 7.6 15.6 B = 10 mg/kg; Dl, 15(q28) C = carboplatin, P = paclitaxel, A = nab-paclitaxel, B =bevacizumab References: Hauschild: Hauschild et al., (2009) J ClinOneal. 27(17): 2823-30 Flaherty: Flaherty et al., (2010) J Clin Oneal.28: 15s (suppl; abstr 8511) N057E: Kottschade et al., (2010) Cancer117(8): 1704-10 N057A: Perez et al., (2009) Cancer 115(1): 119-27 BEAM:Kim et al., (2012) J Clin Oneal. 30(1): 34-41 N0775: Kottschade et al.,(2013) Cancer 119(3): 586-92 Spitler: Boasberg et al., (2011) J ClinOneal. 29 (suppl; abstr 8543)

In the current trial, administration of AB 160 particles is equivalentto a dose of 100 mg/m² of abraxane, and 40 mg/m² of bevacizumab. Theonly study that used BEV and ABX alone was Spitler. Spitler, however,used a higher dose of ABX. The present study also used less than than10% of the dose of BEV reported in previous studies, if the doses areadjusted to the average patient (assumed to have a surface area of 1.9m² and a mass of 90 kg).

Spilter also examined patients who had not been previously treated,while the current study examined patients who had failed previoustreatments. Ineffective prior treatment not only takes time from theexpected PFS, but selects for cancer cells that are more resistant totreatment, and typically leaves a patient in poorer physical condition.Thus, the PFS for a population of patients on a “rescue” therapy (ashere, with AB 160) is expected to have a lower PFS than a naivepopulation. This can be seen in a Phase 2 clinical trial (Hersh et al.,Cancer, January 2010, 116:155) that examined both rescue and naivepatients with Abraxane alone. For previously treated patients withAbraxane alone, the PFS was 3.5 months. Hersh et al. Ann. Oncol 2015,(epub Sep. 26, 2015), reported a 4.8 month PFS for naive patientstreated with ABX alone.

TABLE 9 Performance of AB160 in a limited study against published dataABX dose in BEV dose in Prior average patient average patient PFS Studytreatment (relative dose) (relative dose) (months) AB160 Yes 190mg/patient 76 mg/patient 7.0 (100 mg/m²) (40 mg/m²) Spitler No 285mg/patient 900 mg/patient 8.3 (150 mg/m²) (10 mg/kg) Hersh 2010 Yes 190mg/patient — 3.5 (100 mg/m²) Hersh 2010 No 285 mg/patient — 4.5 (150mg/m²) Hersh 2015 No 285 mg/patient — 4.8 (150 mg/m²)

Thus, early results of the Phase I clinical trial with AB160 indicate anincrease in PFS in late-stage metastatic malignant melanoma inpreviously treated patients. This increase is particularly surprisinggiven that the PFS was greater than those in Spitler, who werechemotherapy naive and were given a higher dose of Abraxane, and analmost 12 fold higher dose of bevacizumab. The dose of BEV used in AB160is far lower than any other study, so the best comparison is notSpitler, but Hersh.

Thus, the ABX/BEV complex (AB160) is superior to sequentialadministration of ABX and BEV, or ABX alone, and achieves this superiorresult with a very low effective dose of BEV. The data is thereforeconsistent with AB 160 having improved targeting of thechemotherapeutics to the tumor, and that this targeting is mediated byBEV. It is possible that the ABX nanoparticle aids in targeting the BEVto the tumor, as albumin is selectively taken up by tumors. It is alsopossible that the existence of the BEV/ABX complex shows greaterstability in vivo than Abraxane.

Example 12 Follow Up Study to Investigate Whether Pretreatment with BEVImproves Targeting

Following the general protocol above, athymic nude mice were injectedwith 1×10⁶ A375 human melanoma cells in the right flank and then treatedwith PBS, 12 mg/kg BEV, 30 mg/kg ABX, AB160, or pretreated with 1.2mg/kg BEV and, 24 hr later, AB160. Data is represented at day 7-post andday 10-post treatment as tumor volume in mm³. F 11A-E track tumor sizeover 10 days. Only mice treated with AB160 (with or without pretreatmentwith BEV) showed a reduction in average tumor volume. See also FIG. 11Fand FIG. 11G.

The day 7-post treatment data, as summarized in FIG. 11F, show thatpretreatment with BEV was associated with a stastically significantreduction in tumor volume over control or BEV alone (p≦0.0001), or ABXalone (p≦0.0001).

The day 10-post treatment data, as summarized in FIG. 11G, again showthat pretreatment with BEV was associated with a stastically significantreduction in tumor volume over control or BEV alone (p≦S0.0001), or ABXalone (p≦0.0001). Pretreatment with BEV before AB160 was also associatedwith a reduction in tumor volume over AB160 alone (p=0.02), withcomplete response in two mice.

In this experiment, a 12 mg/kg dose of BEV was not therapeutic. Theamount of BEV added in the pretreatment group was only 1.2 mg/kg, whichis 1/10 the usual dose in mice. Yet pretreatment with a subtherapeuticdose appears to show improved efficacy of the AB160 nanoparticle. Thisdata support the idea that pretreatment with a subtherapeutic amount ofBEV can clear systemic levels of VEGF, leaving a greater relativeconcentration at the tumor such that tumor-associated VEGF targeting bythe AB160 nanoparticles is more effective.

Example 13 Alternative Means of Delivering Nanoparticles

It is contemplated that nanoparticles of this invention can be directlydelivered to the tumor. For example, nanoparticles can be delivered viaintra-arterial cannula or by direct injection into the turmor. In suchembodiments, it is contemplated that large nanoparticles (e.g., 580 nmor 1130 nm) can be delivered by direct injection into or proximate to atumor.

1. (canceled) 2-30. (canceled)
 31. A lyophilized composition comprisingnanoparticle complexes having an outer surface, wherein each of thenanoparticle complexes comprises: albumin; between about 100 to about1000 cetuximab antibodies, each having a Fc portion and an epidermalgrowth factor receptor (EGFR)-binding portion, wherein EGFR bindingportions of the cetuximab antibodies are arranged on the outer surfaceof the complex; and paclitaxel; said nanoparticle complexes beinglyophilized, and wherein upon reconstitution with an aqueous solutionthe nanoparticle complexes remain capable of binding to EGFR in vivo andfurther wherein said complexes have an average size of from about 130 nmto about 800 nm.
 32. The lyophilized composition of claim 31 that isstable at about 20° C. to about 25° C. for at least 3 months.
 33. Thelyophilized composition of claim 31, wherein each of the nanoparticlecomplexes comprises between about 400 and about 800 antibodies.
 34. Thelyophilized composition of claim 33, wherein less than 0.01% ofnanoparticles in the composition have a size greater than 800 nm. 35.The lyophilized composition of claim 31, wherein said nanoparticlescomplexes have an average size of approximately 160 nm.
 36. Thecomposition of claim 31, wherein the albumin is human serum albumin. 37.The lyophilized composition of claim 31, wherein the antibodies arrangeinto a substantially single layer of antibodies on all or part of thesurface of the nanoparticle complexes.
 38. The lyophilized compositionof claim 31, wherein the nanoparticle having complexes have an averagesize of approximately 160 nm and a dissociation constant between about1×10⁻¹¹ M and about 1×10⁻⁹ M.
 39. A lyophilized composition comprisingnanoparticle complexes having an outer surface, wherein each of thenanoparticle complexes comprises: albumin; between about 100 to about1000 cetuximab antibodies, each having an Fc portion and a EGFR-bindingportion, wherein EGFR-binding portions of the cetuximab antibodies arearranged on the outer surface of the complex; and paclitaxel; saidnanoparticle complexes being lyophilized, and wherein uponreconstitution with an aqueous solution the nanoparticle complexesremain capable of binding to EGFR in vivo and further wherein saidcomplexes have an average size of from about 130 nm to about 800 nm;wherein the lyophilized composition does not contain a bulking agent.